RIBONUCLEIC ACID-POLYPHOSPHATE FROM ALGAE II. PHYSICAL AND CHEMICAL PROPERTIES OF THE ISOLATED COMPLEXES

The properties of RNA-polyphosphate isolated from Anabaena orsynchronously grown Chlorella were examined. Changes in theseproperties at intervals in the life cycle of Chlorella werestudied by the metachromatic reaction for polyphosphate, acid-labilephosphorus, ultraviolet absorption, enzymatic digestion, andcharcoal adsorption. These analyses were made before and afterexhaustive dialysis against distilled water. Before dialysis the polyphosphate gave little metachromaticreaction. Denaturation, induced by dialysis, released the polyphosphatechains for the metachromatic reaction, but the polyphosphatestill was not dialyzable. Dialysis against salt caused no denaturation.Alkaline hydrolysis reduced specific metachrernasy without releasingorthaphosphate. Yeast polyphosphatase destroyed RNA-polyphosphatemetachromasy without releasing much polyphosphate for dialysis.These properties of the RNA-polyphosphate indicate that bothweak bonding and covalent linkages may be involved in the unionof the two substances. Each DEAE-cellulose fraction of RNA-polyphosphate changed inproperties during stages of synchronous Chlorella growth. RNA-polyphosphatein the three areas eluted by highest salt concentration exhibitedthe most striking characteristics for linkage by both weak andcovalent bonds during the first 9-hr of algae growth when thesecomplexes were being synthesized. ¹Journal article number 2909 of the Michigan Agricultural ExperimentStation. Present address: Division of Radiation and Organisms, SmithsonianInstitution, Washington 25, D. C.     

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.     

Some pathways of biosynthesis and degradation of polyphosphates from green algae Acetabularia mediterranea]

The activity of ATP: polyphosphate phosphotransferase was detected in free-cellular extracts of Acetabularia mediterranea. The enzyme activity in cells originally deficient in phosphorus and subsequently transferred into the phosphate-containing medium increases 5-10-fold as compared to normal. Polyphosphate degradation in A. mediterranea is probably produced by polyphosphatase, which was also detected in the free-cellular extract. It was shown that the polyphosphatase activity has two pH optima, i.e. 4.5 and 7.5, and is considerably increased when the cells are transferred into the phosphate-free medium. It is assumed that high-molecular polyphosphates involved in A. Mediterranea metabolism are responsible for regulation of orthophosphate and ATP level in the cells by ATP: polyphosphate phosphotransferase and polyphosphatase.     

Polyphosphate metabolism in the blue-green alga Microcystis aeru-ginosa

The uptake of inorganic phosphorus was studied in an axenicstrain of phosphorus-starved cells of the blue-green alga Microcystisaeruginosa, an organism often causing blooms in freshwater bodies.Rates of growth and of cellular polyphosphate content as a functionof initial orthophosphate in the medium indicate the operationof the ‘phosphorus overplus’ phenomenon in M. aeruginosa,accompanied by formation of volutin granules. The granules wereisolated by a non-aqueous centrifugation method, and identifiedas polyphosphate bodies.     

High molecular mass exopolyphosphatase from the cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene

It has been shown that the high molecular mass exopolyphosphatase localized in cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene. This enzyme is expressed under special culture conditions when stationary phase cells are passing on to new budding on glucose addition and phosphate excess. The enzyme under study releases orthophosphate from the very beginning of polyphosphate hydrolysis.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl₂, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g⁻¹, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.     

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.     

Incorporation of [32P]orthophosphate into long chains of inorganic polyphosphate within lysosomes of human fibroblasts.

When isolated human fibroblast lysosomes are incubated with 4 microM [32P]phosphate at pH 7.0, orthophosphate is transported into lysosomes and is rapidly incorporated into low and high molecular weight products. We have characterized the high molecular weight (HMW) lysosomal material into which [32P]phosphate is incorporated and have found it to consist of long chains of inorganic polyphosphate based on the following observations. 1) greater than 97% of HMW 32P-lysosomal material is converted to [32P]orthophosphate when incubated with 1 N HCl for 20 min at 100 degrees C. 2) Incubation of HMW 32P-lysosomal material at pH 7.0 and 65 degrees C for 96 h results in the formation of [32P]trimetaphosphate, which is known to be produced only from linear chains of polyphosphate under these conditions. 3) HMW 32P-lysosomal material is resistant to degradation by proteinase K, ribonuclease, and deoxyribonuclease and extracts into the aqueous phase during phenol/chloroform extractions. 4) HMW 32P-lysosomal material displays heterogeneous mobility on polyacrylamide gels with most chains ranging in length from 100 to at least 600 phosphate residues. 5) HMW 32P-lysosomal material is partially hydrolyzed under alkaline conditions to yield a continuous ladder of polyphosphate species differing by one or several residues in length on polyacrylamide gels.     

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co²⁺ and Mg²⁺, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co²⁺ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg²⁺ was activated by ADP and arginine but inhibited by ATP.     

EVIDENCE FOR THE OCCURRENCE AND DISTRIBUTION OF INORGANIC POLYPHOSPHATES IN VERTEBRATE TISSUES

Evidence for the occurrence of polyphosphates having apparent chain-lengths ranging from less than 10 to over 5000 orthophosphate units has been found in adult brain as well as in a number of other mammalian tissues which have been examined. There appears to be three times as much polyphosphate in rat brain as there is in rat liver. Adult rat brain appears to contain at least 15 μg of phosphorus as polyphosphate per g of fresh tissue. In addition, neural polyphosphate is extremely labile to catabolic degradation after death whereas hepatic polyphosphate is relatively more stable. The nature of this inorganic polymer was elucidated through use of the technique of ³¹P nuclear magnetic resonance spectroscopy. Further structural evidence was obtained by application of the isotopic dilution technique to ³²P-labelled neural polyphosphate mixed with an abiotically prepared inorganic polyphosphate. The conditions and rates of hydrolysis of the biological polyphosphate and a non-biological polyphosphate were comparable.     

Development of an effective process for utilization of collagen from livestock and fish waste

A procedure for the extraction of protein and production of peptides by enzymic hydrolysis from bone and skin wastes containing collagen was developed. Fat and inorganic components were first removed in a pretreatment step and a high molecular weight protein extracted under acidic conditions (pH 3) using a 1 h reaction time at 60 °C. The molecular weight of extract from pig skin was greater than 100 kDa. The extract had a high water retention capacity, was beneficial for repair of rough skin, had no odor problem and was demonstrated to be safe in skin patch tests. It was thus considered acceptable for use in cosmetic materials. Pretreated fish bone and pig skin were hydrolyzed with a commercial enzyme. The hydrolysates had a high anti-radical activity (IPOX₅₀, 0.18 and 0.45 mg ml⁻¹) and a high potential for decreasing blood pressure (IC₅₀, 0.16 and 0.41 mg ml⁻¹), suggesting the hydrolysates could be a useful additive in food materials.     

Purification of a multiphosphorylated peptide from canine cardiac myosin heavy chains labeled in tissue culture

Partial acid hydrolysis of canine cardiac myosin heavy chains labeled with [³²P]orthophosphate in myocardial cell culture yielded a peptide having a molecular weight between 700 and 1500 and containing phosphothreonine and phosphoserine. The phosphate-rich peptide of myosin heavy chains produced by partial acid hydrolysis was purified first by Sephadex gel filtration, followed by elution with a gradient of formic acid from a Dowex ion-exchange chromatograph. Further identification of the multiphosphorylated peptide was made using high voltage electrophoresis and amino acid analyses. The data described here demonstrate that partial acid hydrolysis (time dependent) can be used to produce partially acid-stable peptides in a good yield.     

Rat liver contains a novel nucleotide, Ypp5'A2'p, related to adenosine 2',5'-bisphosphate

A novel nucleotide, Ypp5'A2'p, has been purified through perchloric acid extraction of rat liver followed by DEAE-cellulose and ion pair high pressure liquid chromatographies. Y stands for an unknown compound, probably a nucleoside, whose sugar moiety is different to beta-D (deoxy) ribose. Treatment of Ypp5'A2'p with snake venom phosphodiesterase renders Yp and adenosine 2',5'-bisphosphate (pAp). After elimination of the terminal phosphate with alkaline phosphatase, the resulting nucleotide (Ypp5'A) yielded Yp and 5'-AMP when hydrolyzed by the phosphodiesterase. The following ultraviolet absorption spectral characteristics were determined at pH 7: Ypp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.79); Yp (lambda max = 279 nm; A250/A260 = 0.70; A280/A260 = 1.70). The molar extinction coefficient found for Yp at 280 nm was 20.6 x 10(3) M-1 cm-1.     

Isolation and certain features of a polyphosphate phosphohydrolase deficient mutant of the fungus Neurospora crassa

A polyphosphatase deficient mutant of Neurospora crassa has been isolated. The criterion for selecting the mutant was the capacity of the fungus to assimilate polyphosphates as the source of exogenous phosphorus. The mutant like the parent strain ad-6, was an adenine auxotroph but differed from the parent strain by a lower growth rate though, at the stationary stage, its biomass reached the same level as in the strain ad-6. The character of changes in the activity of polyphosphatase in the course of growth was the same in the two cultures, but the activity of the enzyme in the mutant was considerably lower at all the growth stages. The content of polyphosphate fractions with the highest molecular weight increased twofold in the mutant culture. These data suggest that there is a close metabolic and topographic correlation between polyphosphatase and the highest molecular weight fractions of polyphosphates in N. crassa.     

CHROMATOGRAPHIC ANALYSES OF ACID-SOLUBLE POLYPHOSPHATES IN CHLORELLA CELLS

Using uniformly ³²P-labeled Chlorella cells as material, compositionof acid-soluble inorganic polyphosphates was studied by paperchromatography and ion-exchange chromatography. 2.By the paper chromatographic analysis it was found that theacid-soluble polyphosphates consisted of highly condensed polyphosphates.Ring-forming tri- and tetrametaphosphates, pyrophosphate andtripolyphosphate were not detected in the acid-soluble fractionof the algal cells. 3.By an ion-exchange chromatography with the use of increasingconcentrations of KCl-solution as eluant, it was found thatthe acid-soluble polyphosphate was a mixture of polyphosphateswith a variety of condensation number (n-values). Polyphosphatesof the n-values between 3 and 15 were only 20% of the totalacid-soluble polyphosphate. The majority of the other polyphospateshad greater n-values which was eluted with 0.5–1.0 M KCl. (Received March 2, 1964; )     

Determination of the role of polyphosphate in transport-coupled phosphorylation in the yeast Saccharomyces cerevisiae

The role of polyphosphate in 2-deoxy-D-glucose transport was studied in yeast cells, pulse-labeled with [³²P]orthophosphate, by comparing the concentrations and specific activities of polyphosphate, orthophosphate and 2-dGlc-phosphate. When 2-dGlc transport was measured under aerobic conditions, it appeared that polyphosphate replenished the orthophosphate pool, indicating that polyphosphate has, at least mainly, an indirect role in sugar phosphorylation. Also in cells with a reduced respiratory capacity, due to a treatment with antimycin A, no direct role for polyphosphate in 2-dGlc transport could be detected. Under these conditions, only a very limited breakdown of polyphosphate occurred, probably because of the small decrease in the orthophosphate concentration.Abbreviations 2-dGlc 2-deoxy-D-glucose - P_i orthophosphate - P_n polyphosphate - SP sugar phosphate     

Polyphosphate Formation from Pyrophosphate in Intact Cells of a Photosynthetic Bacterium, Rhodospirillum rubrum

The hypothesis that the photosynthetic bacterium, Rhodospirillumrubrum, may synthesize polyphosphate using pyrophosphate asa precursor has been tested. The cells were cultured in thepyrophosphate media, and the cell yield, cell shape, and thepolyphosphate content were determined and compared with thatgrown in the orthophosphate media. In the control experiment, the total amount of polyphosphateformed by the cultures grown in the pyrophosphate media wasfound to be up to 70% less than that grown in orthophosphatemedia. When the ATP formation was blocked by using the phosphorylationinhibitor, antimycin A, however, the cells grown in the pyrophosphatemedia formed up to 50% more polyphosphate than that grown inthe orthophosphate media. The results seem to support the hypothesis that Rhodospirillumrubrum may use pyrophosphate as the precursor of polyphosphate. (Received May 26, 1986; Accepted February 21, 1987)     

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.     

5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.