Invertase activity and the kinetin-stimulated enlargement of detached radish cotyledons

Cytokinin treatment is known to promote expansion of light-grown excised radish (Raphanus sativus L. cv Crimson Giant) cotyledons. This expansion, at least in part, seems to be related to an increased accumulation of osmotically active reducing sugars. Kinetin treatment did not cause increased levels of isocitrate lyase activity over the controls, but stimulated increased levels of two invertase forms, designated types I and II. Type I was soluble and type II was insoluble after homogenization in 10 millimolar tris(hydroxymethyl)aminomethane-HCl (pH 7.0). Both types were soluble after homogenization in 300 millimolar NaCl. At low salt concentration, type II was retained on a diethylamioethyl-cellulose column and type I was not. Type II was then eluted from the column at high salt concentration. Types I and II exhibited pH optima of 5.3 and 4.3, Michaelis constants of 4.96 and 1.23 millimolar sucrose, and molecular weights of 65,000 and 57,000 daltons, respectively. The kinetin promotion of reducing sugar accumulation may be related to increased levels of the two invertase forms, but is probably not a result of direct cytokinin-stimulated glyoxysomal activity.     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

Electrophoretic studies on glucosephosphate isomerase and phosphoglucomutase in two types of Anisakis larvae

Agatsuma T. 1982. Electrophoretie studies on glucosephosphate isornerase and phosphoglucomutase in two types of Anisakis larvae. International Journal for Parasitology12: 35–39. Enzyme electrophoresis was carried out between the larval forms. Type I and II, of Anisakis using starch gel. In glucose-phosphate isomerase, considerable polymorphisms were found in each type. At least 5 alleles appear to occur at this enzyme locus in natural populations of both types. Out of 5 alleles, 3 were common to both types. No significant difference was found in their frequencies. However, each larval form can be easily distinguished by the electrophoretic mobility of phosphoglucomutase. It was concluded that enzyme electrophoresis is an alternative useful tool for the identification of larval forms of Anisakis.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Heterogeneity of staphylococcal enterotoxin A on isoelectric focusing and disc electrophoresis.

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N-2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster-moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower-moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.     

Bound TPN as the determinant of polymorphism in methemoglobin reductase

The pyridine nucleotide-dependent, dye-linked methemoglobin reductase from human erythrocytes can be resolved chromatographically or electrophoretically into two principal forms (I and II). Form (II), which migrates more rapidly than (I) toward the positive electrode during electrophoresis at pH 8.3, contains 1 mole of non-covalently bound TPN per mole of protein (MW = 22,000) as judged by spectral characteristics (absorbance maxima at 327 and 340 nm when the protein is treated with cyanide and hydrosulfite, respectively) and by analysis (thin-layer chromatography and coenzymatic activity with isocitrate dehydrogenase) of the nucleotide released by heat denaturation. TPN can also be displaced by treatment of (II) with p-chloromercuriphenyl-sulfonate. Incubation of (I) with TPN at neutral pH results in the formation of (II).     

Separation of three mouse metallothionein isoforms by free-solution capillary electrophoresis

We have used free-solution capillary electrophoresis (FSCE) to separate three distinct mouse metallothionein (MT) isoforms, MT-1, MT-2 and MT-3. FSCE was conducted in an uncoated fused-silica capillary (57 cm × 50 μm I.D., 50 cm to detector) using 50 mM sodium phosphate buffer adjusted to pH 7.0 or 2.0. At neutral pH, each of the three isoform peaks were well resolved from a mixture with the order of migration (MT-1> MT-2> MT-3) related to the net negative charge on the protein. At acidic pH, the migration order was reversed with MT-3 migrating fastest, suggesting MT-3 had a higher net positive charge than MT-2 or MT-1. UV absorbance spectra (190–300 nm) confirmed the presence of Zn in MT-1 and MT-2. MT-3, which was saturated with Cd to stabilize the protein, gave a spectrum characteristic of the Cd---S charge transfer (shoulder at ca. 250 nm). At pH 2.0, the absorbance spectra for all three mouse MTs were characteristic of the metal-free form of the protein (apothionein). Thus, FSCE conducted at neutral pH separates MT isoforms with their metals intact, whereas at pH 2.0, both the Zn and the Cd dissociate from the protein during the run.     

Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg²⁺ and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.Abbreviations AEC adenylate energy charge - DTT dithiothreitol - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FTR ferredoxin-thioredoxin reductase - NADP-MDH NADP-malate dehydrogenase - P glycolate-phosphoglycolate - P glycolate phosphatase-phosphoglycolate phosphatase - PSII photosystem II - PPDK pyruvate, Pi dikinase - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

Diversity and evolution of phycobilisomes in marine <Emphasis Type="Italic">Synechococcus</Emphasis>spp.: a comparative genomics study

Background  

Marine Synechococcus owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrins I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine Synechococcus strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism.     

Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

cAMP-dependent protein kinases, their subunits, and cAMP-binding protein from four sources: a comparison of physical characteristics determined by polyacrylamide gel electrophoresis

The physical characteristics of cAMP-dependent protein kinases and their, regulatory subunits from calf uterus, human uterus, human mammary tumor, and rat pituitary and of cAMP-binding protein from calf uterus were determined by quantitative polyacrylamide gel electrophoresis in buffers containing the detergent, Triton X-100. In the four tissues, protein kinases of either type A¹, with molecular weight (M_r) = 200,000, or type B, of M_r = 80,000, or both, previously described were found. Trivial charge isomerism, or size isomerism, exists within each of the two classes, Protein Kinase A and B. The protein kinase recombined from the regulatory and catalytic subunits is not significantly different from the crude or isolated protein kinase. Protein Kinases A and B exist each in either one of the isozyme forms I and II but these are not reflected in polyacrylamide gel electrophoresis at pH 10.2. Protein Kinase B appears to be a product of the partial proteolysis of Protein Kinase A. The regulatory subunits of Protein Kinases A from the four tissues are distinct from those of Protein Kinases B. No physical distinction exists between regulatory subunits derived from isozyme forms I and II. cAMP-Binding Proteins A and B are physically indistinguishable, by polyacrylamide gel electrophoresis at pH 10.2, from the regulatory subunits of Protein Kinases A and B, respectively.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Fractionation of the proteinases present in the endosperm of germinating seed of Scots pine, Pinus sylvestris

When fresh extracts of endosperms separated from germinating seeds of Scots pine were dialysed at 5°C, proteinase activity on haemoglobin at pH 3.7 showed only a small initial increase, proteinase activities on casein at pH 5.4 and at pH 7.0 increased several-fold, and all the corresponding inhibitor activities disappeared (Salmia and Mikola 1980, Physiol. Plant. 48: 126–130). To find out what happens during dialysis, both fresh and dialysed extracts were fractionated by gel chromatography on Sephacryl S-200. – The fresh extracts had a major proteinase peak (mol. wt. 42,000) with high activity at pH 3.7 and moderate activities at pH 5.4 and 7.0 (pine proteinase I) and a smaller peak (mol. wt. 30,000) with high activity at pH 5.4 and 7.0 and smaller activity at pH 3.7 (pine proteinase II). In dialysed extracts the situation was reversed: the peak of proteinase I was very small while the peak of proteinase II was very high. Apparently, proteinase I is largely inactivated during dialysis while the activity of proteinase II increases, at least partly due to destruction of inhibitors. – The two enzymes were -SH proteinases, as they were completely inhibited by p -hydroxymercuribenzoate; both of them were also inhibited by the endogenous proteinase inhibitors of resting pine seeds. Besides these enzymes, the endosperm extracts contained pepsin-like acid proteinase activity, which is not affected by the endogenous inhibitors. This enzyme activity was largely inactivated during dialysis.     

ζ-Potential and surface charge of Thermoplasma acidophila

The surface charge density and the ζ-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca²⁺ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the ζ-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm^-2 which corresponds to one elementary charge per 3500 A².     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

Hydroxyapatite chromatography of phage-display virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.     

Studies on interaction of oligopeptides with sodium dodecyl sulfate: Stopped-flow kinetics of chemical modification of tryptophan residues withN-bromosuccinimide

The stopped-flow kinetics of the reaction between oligopeptides containing tryptophan residues andN-bromosuccinimide (NBS) were studied in 50 mM sodium phosphate buffer (pH 7.0) containing sodium dodecyl sulfate (SDS). Decreases in the reaction rates attributable to the interaction between oligopeptides and SDS were observed, and oligopeptides studied were classified into types I and II on the basis of the interaction modes. Type I oligopeptides were dissolved in SDS micelles; type II oligopeptides interacted cooperatively with SDS monomers. The manner of interaction between SDS and oligopeptides of type II could be interpreted by a simple equilibrium relation: oligopeptide+n·(SDS)=oligopeptide·(SDS) _n .     

Multiple types of cell surface heparan sulfate are produced by primary cultures of embryonic mouse cells

The cell surface heparan sulfate produced by primary cultures of 12-day mouse embryo cells has been found to consist of at least two forms, designated I and II. These two forms can be distinguished by both ion-exchange chromatography on DEAE-cellulose and eletrophoresis at pH 1. However, no difference in molecular weight is observed when the two forms are compared by gel filtration on Bio-Gel A-15m. These data suggest that the two forms differ in their content of sulfate residues. Multiple types of cell surface heparan sulfate are also produced by primary cell cultures derived from various mouse embryonic organs, including heart, lung, kidney and liver. Type II, the minor form produced by the primary embryonic mouse cells, behaves on ion-exchange chromatography and electrophoresis at pH 1 as the heparan sulfate produced by several mouse cell lines that exhibit contact inhibition of growth. The predominant form, type I, behaves on ion-exchange chromatography as the heparan sulfate derived from either DNA or RNA virus-transformed cell lines which lack growth control. The cell surface heparan sulfate produced by chick myoblasts, human fibroblasts, and bovine endothelial cells behave as single types on ion-exchange chromatography. These data suggest that an individual cell type produces a single type of cell surface heparan sulfate and provide support for a model in which cell-cell interactions are mediated, in part, by the quantity and, possibly, arrangement of sulfate residues within the heparan sulfate polymer.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate.Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid would be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8 and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydroflate, lower affinity of N⁵-methyl-tetrahydrofolate, and no apparent affinity for N⁵-formytetrahydrofolate or methotrexate.