Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

In situ determinations of bacterial selectivity and filtration rates by five cladoceran zooplankters in a hypertrophic subtropical reservoir

Cladoceran in situ feeding rates on natural bacteria labelledwith [methyl-³H] were studied in parallel with feeding ratedeterminations on ¹⁴C-labelled Chlorella in a hypertrophic subtropicalreservoir (Lake Hartbeespoort) through spring and summer (1986/87).Community filtration rates (CFR5) on bacteria and algae weresimilar, but selection for Chlorella (relative to natural bacteria)increased from midsummer in association with declining bacterialdensity and increasing dominance of ‘inedible’ componentsof the natural phytoplankton. Species-specific filtration rates(SSFRs) were determined for Daphnia pulexllongispina, Ceriodaphniareticulata, Diaphanosoma excisum, Bosmina longirostris and Moinamicrura during their respective seasonal occurrence in the studyperiod. SSFRs on algae and bacteria increased with body length(L, mm) in all species apart from Bosmina. Species-specificdifferences in absolute feeding rate (FR, ml animal^–1day^–1), the slope of the FR-L relationship and bacteriaselectivity were evident. The feeding rate of all cladoceranson bacteria is described by the power equation FR 5.231L^1.42FR values on bacteria relative to FR values on algae averaged     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

Continuous plankton records stand the test of time: evaluation of flow rates, clogging and the continuity of the CPR time-series

The Continuous Plankton Recorder (CPR) survey is one of themost extensive biological time-series in existence and has beenin operation over major regions of the North Atlantic since1932. However, there is little information about the volumeof water filtered through each sample, but rather a generalassumption has persisted that each sample represents 3 m³. Datafrom electromagnetic flowmeters, deployed on CPRs between 1995and 1998, was examined. The mean volume filtered through sampleswas 3.11 m³ and the effect of clogging on filtration efficiencieswas not great. Consequently, even when the likely variationsin flow due to clogging are taken into account, previously identifiedlinks between zooplankton abundance and climatic signals remainstrong.     

Stop-flow studies of distribution of filtration in rat lungs

Lin, W., E. Jacobs, R. M. Schapira, K. Presberg, andR. M. Effros. Stop-flow studies of distribution offiltration in rat lungs. J. Appl.Physiol. 84(1): 47-52, 1998.The stop-flow approach was used to investigate where filtration occurs in the pulmonary vasculature after elevation of left atrial pressure andaspiration of HCl. Rat lungs were perfused for 11 min at zero leftatrial pressures, and then flow was stopped for 10 min and left atrialpressures were increased to 20 cmH₂O. Thereafter, ³HOH was instilled into the airspaces, and the pulmonary vasculature was flushed by perfusing it fromthe pulmonary artery to left atrium (anterograde flush) or in theopposite direction (retrograde flush). Increases in fluoresceinisothiocyanate (FITC)-dextran (molecular weight 2,000,000) indicatedfiltration, and these preceded increases in³HOH after anterograde but notretrograde flushes. This suggests that some filtration occurred throughvessels that were relatively venous compared with those through which³HOH exchange had occurred.Filtration increased fivefold after instillation of 0.1 N HCl inisotonic saline into the air spaces before perfusion. Increases inEvans blue-labeled albumin concentrations were <40% those ofFITC-dextran, indicating loss from the vasculature, but increases inunlabeled albumin and FITC-albumin were comparable.

     

Experimental studies into the feeding biology of rotifers in brackish water

Mass developments of rotifers of the genus Brachionus, and especiallyof B.quadridentatus, occur regularly in the largely hypertrophicchain of shallow waters (‘boddens’) south of theDarss-Zingst peninsula (Southern Baltic). Interest in the autecologyof the species is, therefore, considerable. Various food sourceswere used in laboratory experiments to ascertain the food requirementsof B.quadridentatus, determine its filtration and ingestionrates, and assess its food particle size-selection ability.Growth experiments showed that the chlorophyceans Nannochlorissp. and Chlorella vulgaris possess considerable nutritionalvalue for the species, whereas abundances declined when Microcystisfirma, detritus from Enteromorpha sp. and only bacteria (Pseudomonas),respectively, were provided as food sources. Filtration ratesvaried between 0.02 and 1.73 µl ind.^–1 h^–1,and ingestion rates between 121 and 5560 cells ind.^–1h^–1, depending on the filtration rate and algal concentration.Investigations into food particle size selection using fluorescentlatex particles revealed that particle size influences foodparticle intake. When particles of different sizes were mixed,the animals showed a preference for the larger particles andingested the smaller ones with a diameter of 1–2 µmless efficiently. The brackish water species Brachionus plicatiliswas studied besides B.quadridentatus in all experiments. Theformer species proved to be superior both in its range of utilizableparticle sizes and its growth rate. The experiments with laboratorycultures were backed up by studies performed with various rotiferspecies taken from natural populations.     

Uptake of [32P]orthophosphate by algae adn bacteria in Lake Kinneret

Differential filtration was used to apportion [³²p]orthophosphate(P₁) uptake to predominantly bacterial (<3 µm) or algal(>3 µm) components of Lake Kinneret microplankton.Bacteria generally showed preferential ³²P_i uptake in comparisonwith algae. Nevertheless, in most cases, the relative proportionof ³²P counts retained on 3 µm filters was greater thanthe proportion of ¹⁴C counts from heterotrophic bacterial incorporationof [¹⁴Clglucose, indicating that algae were competing for P_iwith bacteria with some measure of success. Most time courseexperiments did not show any consistent transfer of ³²P frombacteria to algae. The addition of a bacterial inhibitor (garamycin)caused a relative increase in the proportion of algal to bacterial³²P_i uptake. Added organic P substrates lowered the amount of³²P_i uptake and appeared to be preferentially utilized by bacteria.Apparent residence times for P_i in Lake Kinneret ranged from0.4 h (prior to overturn) to 17.4 h during bomothermy. Despitelow ambient P_i concentrations, P limitation in Lake Kinneretis not as extreme as in many other aquatic environments.     

Natural filtration rates of zooplankton in a closed system: the derivation of a community grazing index

Measurements of individual filtration rates by natural suspensionsof zooplankton are presented. The products filtration rate xanimal concentration, calculated for each of several species-sizecategories, are summated to estimate the total volume of waterfiltered by the zooplankton per unit time, here defined as the"Community Grazing Index" (CGI). Seasonally, CGI varies overnearly three orders of magnitude. The Daphnia hyalina individualspresent frequently contributed >90% of CGI. Individual filtrationrates were highest when the concentrations of suitable foodswere limiting (equivalent to 0.1–0.2 µg C ml^–1),but were depressed after long (3-week) periods of low food availabilityand when large Microcystis colonies dominated the phytoplankton.Approximately one order of magnitude separated these extremes.Fluctuations in CGI owe relatively more to change in the concentration,size and species distribution of the animal population thanto changes in individual filtration rate. ¹Present address: Anglian Water Authority, Oundle, PeterboroughPE8 4AS, UK     

Methods of enhancing the recovery of plasmid genes from neutralised cell lysate

In this study we have investigated the use of flotation and filtration, singly and combined, to enhance the separation of plasmid containing liquors from neutralised lysates with very different levels of solids. Filtration of crude neutralised lysates, containing roughly 100 g l^у solids, through various diatomaceous earth and cellulose precoat materials was invariably accompanied by severe loss of plasmid through adsorption and/or absorption. The use of more refined and inert filter aids did not alleviate these problems. The finest filter aid, Celatom FP-1SL, gave the best compromise of filtrate clarity (solids content of 0.05 g l^у) and plasmid purity (71%) and was selected for further studies involving combined use of flotation and filtration. Removing the vast bulk of solids prior to filtration by flotation of the floc and draining of the plasmid liquor beneath, impacted dramatically on the filtration performance. Though systematic reductions in the solids challenge per unit filter area were accompanied by increased flux, elevated levels of solids extrusion, chromosomal DNA and protein contamination were also observed, and losses of plasmid to filter aids were still high. We have observed that increasing the scale of operation during lysis and neutralisation from 0.3 or 0.6 l to 15 l is accompanied by significant improvements in separation of cell debris solids from the plasmid and increased recoveries of the plasmid containing liquor. At the latter scale, the drained liquor contained ~80% of the plasmid and the solids content was only 0.2 g l^у.     

Isolation, Purification and Some Properties of Cytochrome c-551 from Chromatium vinosum

Cytochrome c-551 was isolated and purified from a photosyntheticbacterium Chromatium vinosum by ammonium sulfate fractionation,ion-exchange chromatography and gel filtration. The cytochromehad absorption maxima at 280, 407 and 523–524 nm in theoxidized form, and 416, 521 and 549.5 nm in the reduced form.The reduced-minusoxidized difference millimolar absorption coefficientwas 9.90 mM^–1cm^–1 for the wavelength pair, 550.5minus 540 nm. The molecular weight of the cytochrome was 16,000by gel filtration on Sephadex G-100 and 15,500 by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The midpoint redox potential was +240 mVat pH 8.0. Cytochrome c-551 was released from bacterial cells when spheroplastswere produced but EDTA and lysozyme treatments. The releasedcytochrome had the same properties as those of the cytochromepreparation obtained by disruption of cells through a Frenchpressure cell. This confirms the earlier suggestion that cytochromec-551 is located in the periplasmic space of cells. (Received August 21, 1982; Accepted October 28, 1982)     

Isolation and some properties of copper-binding proteins found in a copper-resistant strain of yeast

Copper-binding proteins were extracted from a copper-resistantstrain of Saccharomyces cerevisiae which was obtained by repeatedsubculturing in a copper-containing medium. They were separatedinto three types through purification steps such as salt fractionation,gel filtration and preparative polyacrylamide gel electrophoresis.They resembled each other in amino acid composition. Acidicamino acids, lysine, serine, glycine and half-cystine constituteda large part of the protein, with a small amount of hydrophobicamino acids. Aromatic amino acids and methionine were almostabsent. The molecular weight of the components was estimatedto be about 10,000 by Sephadex gel filtration and electrophoresison polyacrylamide gel (slope method). Absorption spectra ofthe components exhibited a broad band at 275 nm, but none inthe visible region, thus resembling that of copper-thionein.Moreover, the absorption band at 275 nm changed markedly onaddition of Ag⁺, Hg2⁺, CN^– or H₂O₂, which are well knownas thiol reagents. These components were abo produced in theparent cells, if they could grow in a copper-containing medium.Based the results of experiments using various culture conditionsand some other yeast species, a possible role of the componentsis discussed. (Received July 13, 1976; )     

Nutrient cycling through phytoplankton, bacteria and protozoa, in selectively filtered Lake Vechten water

The breakdown of organic carbon of dead Synechococcus cell walls,added to selectively filtered Lake Vechten water, was not acceleratedby protozoa. During 4 weeks of incubation at 15°C no significantdecrease of total organic carbon was observed. However, heterotrophicnanoflagel-lates (HNAN) and ciliates strongly increased theremineralization of N and especially P, from both cell wallsand cell extract. Bacterioplankton growth did not result innet P mineralization but in P uptake. P was remineralized onlyin the presence of protozoan grazers. Both HNAN and ciliatesgrazed on bacteria, with ingestion rates estimated at 27–96bad HNAN^–1 h^–1 and 129 bact ciliate^–1 h^–1respectively. Grazers increased N mineralization too, althoughN was also mineralized in the absence of protozoa. The phytoplanktoncell walls yielded less P but more N remineralization than thecell extract. Thus, protozoa can strongly accelerate cyclingof specific nutrients through plankton. Nuclepore filters werefound to cause artificial DOC release during selective filtration.     

Purification and Properties of Soluble Chlorophyllase from Tea Leaf Sprouts

Soluble chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1.14 [EC] ) was purified 650-fold from tea leaf sprouts byammonium sulfate fractionation and gel filtration through SephadexG-200 and Sepharose CL-6B. The purified enzyme showed two bandson polyacrylamide gel electrophoresis and the specific activitywas 2.6 µmol chlorophyll a hydrolyzed min^–1 mg^–1of protein. The molecular weights determined by Sepharose CL-6Bwere 910,000 and 350,000, indicating high molecular aggregates.The subunit molecular weight estimated by sodium lauryl sulfate-polyacrylamidegel electrophoresis was 38,000. The isoelectric point was 3.9.The optimum pH was 5.5 in acetate buffer and the Km value forchlorophyll a was 10 µM. This enzyme did not require athiol compound nor metal ion such as Mg²⁺. (Received January 26, 1981; Accepted April 3, 1981)     

Aspects of the feeding ecology of turbid water zooplankton. In situ studies of community filtration rates in silt-laden Lake le Roux, Orange River, South Africa

Diel vertical studies of zooplankton community filtration rates(CFR) were undertaken in situ over two annual cycles in a shelteredbay of Lake le Roux, a large silt-laden oligotrophic reservoirin the arid subtropics of South Africa. The grazer communitywas dominated by a copepod, Meradiaptomus meridianus, whileDaphnia gibba. D. barbara and Moina brachiara accounted forthe balance. Spot estimates of CFR varied from 0.1 to 75% d^–1( = 12.2) in the upper 10 m, while depth-integrated values ranged seasonally from 0.1 to 15% d^–1 ( = 7.4). Most variation in CFR was attributable toseasonal changes in grazer biomass (0–408, = 58.2 µg 1^–1 or 3–1000, 360 mg m^–2 dry wt), and temperature (12–22C). Thesevariables are used to construct multiple linear regression modelsfor the prediction of CFR in this system. Some higher CFR values(up to 260% d^–1 were measured in wanner (up to 26.5C)surface waters with an unusually rich zooplankion (786 rg 1^–1).No significant did vertical changes in CFR or grazer biomasswere observed. Both variables declined sharply with depth. Inorganicturbidity dominated the seston, and algal carbon probably accountedon average for less than about 20% of the total POC available.The feeding responses of this turbid-water community were generallyconsistent with observations made on other assemblages, apartfrom seemingly high specific filtration rates which, atypically,were inversely related to temperature.     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.     

Particulate and dissolved phytoplankton production of the Patos Lagoon estuary, southern Brazil: comparison of methods and influencing factors

Uptake rates of ¹⁴C (filtration and the acidification-bubblingmethod—ABM) were measured weekly in a shallow region ofthe Patos Lagoon estuary (3207'S, 5206'W) between March 1989and March 1990. Phytoplankton production varied seasonally,the lowest values occurring in the austral winter (June–August1989) and the highest rates during spring and summer (March1989; September 1989–March 1990). Particulate carbon productionvaried between 0.65 and 70.6 mg C m^–3 h^–1 and wasmostly associated with organisms <20 µm (mean = 73.4%).Dissolved organic carbon (DOC) released by phytoplankton variedbetween 0.1 and 89.3 mg C m^–3 h^–1 representing     

Purification and general properties of spinach leaf nitrite reductase

Nitrite reductase was isolated from spinach leaves. The enzymewas purified 168-fold by a procedure involving extraction withphosphate buffer, gel filtration on Sephadex G-200, ion-exchangechromtography on DEAE-Sephadex A-50, and adsorption on hydroxyapatite.The preparation was homogeneous in the ultracentrifuge withsedimentation coefficient at infinite dilution (s?_20,w) of 4.57S. Disc electrophoresis revealed some small bands together witha major protein band. The molecular weight of the spinach nitritereductase was estimated to be 60,000 by gel filtration on SephadexG-100 while a molecular weight of 72,000 was obtained from thesedimentation-diffusion coefficients of the protein. Resultsof sodium dodecyl sulfate gel electrophoresis suggested thatthe enzyme molecule consists of two subunits of molecular sizeof 37,000. After close examination of assay systems based onsodium dithioniteviologen dye procedures, we developed a moreelaborate, improved chemical assay method. Some enzymatic propertiesof the purified nitrite reductase were examined. ¹This work was reported in part at the Annual Meeting of JapaneseSociety of Plant Physiologists, April 6–8, 1972. (Received November 16, 1972; )     

Artificial Insemination 'Regulated by EDTA' in the Monoecious Brown Alga Fucus evanescens

As part of an attempt to control the fertilization of a monoeciousbrown alga Fucusevanescens, the effects of EDTA on the releaseand fertilization of gametes were studied. When receptacleswere treated for liberation of gametes by soaking in plain seawater,egg-packets and spermatophores were discharged from oogoniaand antheridia, respectively, with the gametes retained withintheir packing envelopes. After a while, the envelopes were disruptedand the gametes were released. Thirty min after the soaking,over 99% of eggs were fertilized. However, when receptacleswere soaked in seawater that contained 0.5 mg m1^–1 Na₂.EDTAat 4°C, the release and fertilization of gametes were preventedafter egg-packets and spermatophores had been released fromthe receptacles. Release of gametes from such egg-packets andspermatophores occurred rapidly when the medium was dilutedwith an excess of plain seawater. The chelating agent affectedthe disruption of egg-packets and spermatophores, but it didnot affect the subsequent fertilization and development of thefertilized eggs. On the basis of these results, normal unfertilizedeggs and sperm were isolated separately by filtration and centrifugationof the released packing envelopes in the presence of 0.5 mgml^–1 Na₂EDTA at 4°C. Artificial insemination usingthe isolated gametes was successful. ³ Present address: Akan Board of Education, Akan, 085-02 Japan.     

Purification and characterization of {delta}-aminolevulinic acid dehydratase from Chlorella regularis

-Aminolevulinic acid dehydratase (-aminolevulinic acid hydrolyaseEC 4.2.1.24 [EC] ) which catalyzes the formation ofporphobilinogenfrom two molecules of -aminolevulinic acid (ALA) was purifiedfrom Chlorella regularis 737-fold by acetone and ammonium sulfatefractionations, DEAE-cellulose column chromatography, and SephadexG-200 gel filtration. The enzyme had an optimum pH of 8.5 inTris-HCl buffer and required either Mg²⁺ or Mn²⁺ for its maximumactivity. The Km values for Mg²⁺, Mn²⁺ and ALA were 15 µM,10µM, and 0.5 mM, respectively. The enzyme was not activatedby thiol compounds, but was inhibited by p-chloromercuribenzoate.The molecular weight estimated by gel filtration was 316,000and the isoelectric point was 5.25. (Received October 18, 1978; )     

On the radiocarbon method: filtration or the acidification and bubbling method?

It has recently been claimed that ¹⁴C fixation measured by theacidification and bubbling method gives considerably highervalues than using the filtration method. The observed discrepancyis probably due to a particle fraction (<0.8 >0.2 µm)not included in those results. The size fraction <1.0 >0.2µm can be a significant fraction of the ¹⁴C-fixation especiallyin oligotrophic water. The ¹⁴C-activity in that size fractionis probably due to bacteria assimilating labelled extracellularproducts.