The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

The origin of Q-independent derivatives of phage lambda

Summary qsr (Q-independent) phages are characterised by the replacement of the region of the genome that contains Q, S, R, and the late gene promoter, P_R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that p4 and qin A 3, two such Q-independent phages, are the product of recombination between and a defective lambdoid prophage (the qsr prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K 12 strain AB1157 from which qsr phages cannot be generated, the qsr prophage has suffered an internal deletion. That the qsr prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.     

The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EB extraction buffer - EGTA ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - TBS Tris buffered saline - TEB Tris-EGTA buffer     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Conformational dynamics in mixed alpha/beta-oligonucleotides containing polarity reversals: a molecular dynamics study using time-averaged restraints

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3-3 and 5-5 phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two -containing duplexes – d(GCGAAT-3-3-T-5-5-CGC)₂ and d(ATGG-3-3-T-5-5-GCTC)r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (d _av, J _rms, and J _av) and improvements in sixth-root R-factors (R ^x). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R ^x values. In both systems, the backbone torsion angles comprising the 3-3 linkage as well as the (high S-) sugars of the -nucleotide and preceding residue (–1) are relatively static, while the conformations of the 5-5 linkage and the sugar in the neighboring -nucleotide (+1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.     

Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

Pre-moult foraging trips and moult locations of Emperor penguins at the Mawson Coast

The movements of nine breeding adult emperor penguins Aptenodytes forsteri from two colonies, Auster (67° 23S 64° 04E) and Taylor Glacier (67° 28S 60° 54E), were determined by satellite telemetry on their pre-moult foraging trips. While preparing for their annual moult the penguins travelled for 22–38 days and reached distances of up to 618 km from the colony. Six of the nine tracked penguins were followed to three different moult locations all to the west of their breeding colonies and near other known emperor penguins colonies, such as Kloa Point (66°38S, 59°23E) and Fold Island (67°17S, 59°23E). Sea-ice conditions changed throughout the tracking period; as the birds travelled north the sea-ice contracted south.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

A New Aerobic Gram-Positive Bacterium with a Unique Ability to Degrade ortho- and para-chlorinated Biphenyls

Strain B51 capable of degrading polychlorinated biphenyls (PCB) was isolated from soil contaminated with wastes from the chemical industry. Based on its morphological and chemotaxonomic characteristics, the strain was identified as a Microbacterium sp. Experiments with washed cells showed that strain B51 is able to degrade ortho- and para-substituted mono-, di-, and trichlorinated biphenyls (MCB, DCB, and TCB, respectively). Unlike the known PCB degraders, Microbacterium sp. B51 is able to oxidize the ortho-chlorinated ring of 2,2-DCB and 2,4-DCB and the para-chlorinated ring of 4.4-DCB. The degradation of 2,4-DCB and 4,4-DCB was associated with the accumulation of 4-chlorobenzoic acid (4-CBA) in the medium in amounts comprising 80–90% of the theoretical yield. The strain was able to utilize 2-MCB, 2,2-DCB, and their intermediate 2-CBA and to oxidize the mono(ortho)-chlorinated ring of 2,4,2-TCB and the di(ortho-para)-chlorinated ring of 2,4,4-TCB. A mixed culture of Microbacterium sp. B51 and the 4-CBA-degrading bacterium Arthrobacter sp. H5 was found to grow well on 1 g/l 2,4-DCB as the sole source of carbon and energy.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol