Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Summary Sphaerotilus natans was shown to have a fourfold lower K _mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K _mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.Offprint requests to: C. F. Kulpa Jr     

The ovarial enzymes hydrolysing l-leucyl- and dl-alanyl-β-naphthylamide

Summary Fractionation of the rat ovarial tissue homogenate was performed using gel filtration on Sephadex G 200 and by starch gel electrophoresis. The activities hydrolysing l-leucyl--naphthylamide (Leu--NA) and dl-alanyl--naphthylamide (Ala--NA) were determined and partially characterized. Leu--NA was hydrolysed by four separate enzyme activities separated by both methods. Two of them were thiol-activated, one metal-activated and inhibited by EDTA. One was affected by neither metal chelators nor by sulfhydryl reagents. Ala--NA was hydrolysed by the three first-mentioned activities, but not by the last one. In addition, Ala--NA was hydrolysed by two other activities which were totally inhibited by metal chelators. These were clearly separated only using starch gel electrophoresis. The possibilities for the histochemical demonstration of these activities are discussed.     

Structural and Mechanistic Studies of a Stabilized Subunit Dimer Variant of Escherichia coli Bacterioferritin Identify Residues Required for Core Formation

Bacterioferritin (BFR) is a bacterial member of the ferritin family that functions in iron metabolism and protects against oxidative stress. BFR differs from the mammalian protein in that it is comprised of 24 identical subunits and is able to bind 12 equivalents of heme at sites located between adjacent pairs of subunits. The mechanism by which iron enters the protein to form the dinuclear (ferroxidase) catalytic site present in every subunit and the mineralized iron core housed within the 24-mer is not well understood. To address this issue, the properties of a catalytically functional assembly variant (E128R/E135R) of Escherichia coli BFR are characterized by a combination of crystallography, site-directed mutagenesis, and kinetics. The three-dimensional structure of the protein (1.8 Å resolution) includes two ethylene glycol molecules located on either side of the dinuclear iron site. One of these ethylene glycol molecules is integrated into the surface of the protein that would normally be exposed to solvent, and the other is integrated into the surface of the protein that would normally face the iron core where it is surrounded by the anionic residues Glu⁴⁷, Asp⁵⁰, and Asp¹²⁶. We propose that the sites occupied by these ethylene glycol molecules define regions where iron interacts with the protein, and, in keeping with this proposal, ferroxidase activity decreases significantly when they are replaced with the corresponding amides.Bacterioferritin (BFR)⁴ is a prokaryotic form of ferritin that has been identified in a number of bacteria (1–3). Despite low sequence similarity with eukaryotic ferritins, the three-dimensional structures and functional properties of BFRs from Escherichia coli, Rhodobacter capsulatus, Desulfovibrio desulfuricans, and Azotobacter vinelandii (4–10) are remarkably reminiscent of those reported for mammalian ferritins. For example, BFRs are oligomeric proteins comprised of 24 subunits (∼18 kDa each) that catalyze oxidation of Fe²⁺ by dioxygen (ferroxidase activity) to promote formation of a mineralized iron core that can contain as many as 2700 iron atoms/ 24-meric molecule (11). On the other hand, those BFRs that have been characterized differ from the mammalian proteins in that the 24 subunits are identical, and each possesses a catalytic dinuclear iron center that is referred to as the ferroxidase site (in mammalian ferritins, only the H-chains possess such catalytic sites). The pairwise arrangement of BFR monomers within the 24-mer creates 12 binding sites for heme, commonly protoheme IX but iron-coproporphyrin III in D. desulfuricans BFR, in which a methionyl residue on the surface of adjacent BFR monomers provides an axial ligand to create a b-type heme-binding site with bismethionine axial coordination (12, 13). Although a functional role for the heme of BFR has not been identified, the functional role of BFR is believed to be in iron storage and detoxification (14), thereby protecting against oxidative stress (15).The subunits of BFR are arranged to form eight 3-fold channels and six 4-fold channels. These channels have been proposed as possible entry and exit routes for iron incorporation into or release from the central iron core. For human ferritin, the 3-fold channel plays a significant role in the transport of iron into the iron core (16), but a similar role for this channel in BFR has not been demonstrated.The dinuclear ferroxidase site located within each subunit binds two iron atoms. Coordination of these iron atoms involves Glu⁵¹ and Glu¹²⁷ as bridging ligands for both irons, Glu¹⁸ and His⁵⁴ as ligands for FE1, and Glu⁹⁴ and His¹³⁰ as ligands for FE2. Previous studies of E. coli BFR have demonstrated that the ferroxidase center is essential for core formation and that core formation involves at least three kinetically distinguishable phases (11, 17). Phase 1 involves the very rapid reversible binding of two Fe²⁺ ions to each of the 24 dinuclear ferroxidase centers and can be studied by monitoring small changes in the spectrum of the bound heme. Phase 2 occurs in the presence of dioxygen (or an alternative oxidant such as hydrogen peroxide) and involves the rapid oxidation of each di-Fe²⁺ center to form an intermediate that is probably an oxo- or hydroxo-bridged di-Fe³⁺ center. In the presence of Fe²⁺ exceeding the amount required to saturate the ferroxidase centers, a slower reaction, Phase 3, is observed in which a large ferric oxyhydroxo mineral is synthesized within the protein cavity. The change in absorbance at 340 nm that is observed during aerobic addition of Fe²⁺ to apo-BFR results from Phases 2 and 3 but is influenced by the kinetics of Phase 1. Although Phases 1 and 2 are well characterized, less is known about Phase 3. This phase probably involves the interaction of Fe²⁺ (or Fe³⁺) with amino acid residues on the inner surface of the ferritin oligomer, as part of a complex and poorly defined process known as nucleation. Further information on this phase of core formation is now required.An assembly variant of E. coli BFR (E128R/E135R) has been shown previously to form stable subunit dimers that bind one equivalent of protoheme IX and not to form higher order oligomers (18). Each monomer in this minimal functional unit can form a dinuclear iron center that catalyzes the formation of a minimal iron core comprised of four to six iron atoms before precipitating (18, 19). The overall kinetics of Fe²⁺ oxidation observed on addition of Fe²⁺ to this variant are similar to those observed for wild-type BFR but have not been reported in detail. Nevertheless, the properties of this variant are of interest because the minimal structural unit that it forms constitutes a potentially important experimental model for evaluating detailed mechanistic features of BFR function. Simplification of the oligomeric structure of the protein as represented by this variant form of BFR makes the inner surface of the protein as accessible to bulk solvent as the outer surface, thereby removing any kinetic influences of the channels present in the 24-mer protein.The present paper reports detailed kinetic and structural studies that validate this dimeric variant of BFR as a model of the minimal functional unit of wild-type BFR. In addition, the crystallographic structural data suggest a likely functional role of acidic inner surface residues in iron core formation that led to construction of a family of variants of the stable subunit dimer involving replacement of Glu⁴⁷, Asp⁵⁰, and Asp¹²⁶ by site-directed mutagenesis. Kinetic studies of these additional variants confirm a functional role for these residues and lead to the proposal of a model of BFR action.     

Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10^–3 min^–1 at 120 min after the beginning of the washing period and fell slowly (9% hr^–1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 m lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.     

Lipopolysaccharide of Providencia rettgeri

The chemical constitutional analysis of the lipopolysaccharide (LPS) isolated from Providencia rettgeri was carried out. Polyacrylamide gel electrophoresis using sodium dodecylsulfate or sodium deoxycholate showed that the lipopolysaccharide mostly consisted of short sugar chains. The lipid A was precipitated out after mild acid hydrolysis of LPS. From the supernatant degraded polysaccharide and unsubstituted core fractions were isolated. Compositional analysis of the core material revealed the presence of galacturonic acid, galactose, glucose, glucosamine, l-glycero-d-manno-heptose, 3-deoxy-d-manno-octulosonic acid, alanine and phosphorus. Methylation analysis of the core material indicated the presence of terminal units of glucose, galacturonic acid and glucosamine. The chemical structure of the lipid A was elucidated. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester and glycosidically-bond phosphate residues. The ester-bound phosphate was found to be substituted by a 4-amino-4-deoxy-l-arabinosyl residue. The amino groups of the backbone disaccharide are N-acylated by 3-O-(14:0)14:0 and 3-O-14:0.Two hydroxyl groups of the disaccharide are esterified by 3-O-(14:0)14:0 and 3-O-14:0. The taxonomical importance of these structural details will be discussed.Abbreviations LPS lipopolysaccharide - l-d-heptose l-glycero-d-manno-heptose - dOclA 3-deoxy-d-manno-octulosonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - PS degraded polysaccharide - glc-ms combined gas liquid chromatography-mass spectrometry     

Ferritin—A general metal detoxicant

Binding of nonferrous metal ions to ferritin was compared to that of the phosphate-free or phosphate containing synthetic iron cores. The Scatchard plots for the synthetic cores reveal a high affinity site for Cd, Zn, Be, and Al, with K_D in the range 10^?5–10^?7 M. Preloading the cores with phosphate increased the number of metal ions bound without altering the K_D. The metal ions with smaller ionic radii (Be, Al) were bound in larger numbers than those with larger ionic radii (Cd, Zn). Ferritin isolated from soybean (Glycina max), horse spleen, and rat liver bound the metal ions in amounts larger than predicted from their iron core. Whereas the iron cores and their nonferrous metal ion complexes were insoluble, those in the protein shell remained in solution. Thus apoferritin precipitated with lower concentrations of aluminum than did holoferritin. Also, Al bound to apoferritin reduced the rate of iron loading into the protein.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H₂ and CO₂: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. I. Formation of secondary lysogens

Summary The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The phage DNA failed to get inserted at the secondary site(s) of the gal operon (frequency <2.6x10^-8) in the presence of the primary (normal) att site. By contrast, 80 and the att80 hybrid (x80) became integrated into wild-type Escherichia coli at at least two secondary att sites of the btuB locus, and the latter near purE and purC as well (frequency 2x10^-3-10^-4). The integration of 80 and att80 into btuB occurred with about the same frequency as in cells in which the normal insertion site had been deleted (0.7-4.0x10^-6). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found at the primary att site. We also failed to observe the integration into other loci of 80 and att80 with the formation of secondary monolysogens (frequency <0.0035 at MOI-10^-3 or 10). It is presumed that these prophages become integrated at secondary att sites only if the primary site is occupied.Abbreviations MOI multiplicity of infection (PFU/cell) - PFU plaque-forming unit - TP transducing phage - P1/HfrH P1vir multiplied on HfrH - Rif-R rifampin-resistance - Int int protein     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Involvement of Mhc Loci in immune responses that are not Ir-gene-controlled

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymphnode cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative response of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time -antibodies specific for the A (AA) or E (EE) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastoinyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Irr genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of Ir-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by Ir genes: both types involve the recognition of the antigen, in the context of Mhc molecules.Abbreviations used in this paper A class II MHC molecule consisting of the A and A chains - ADH alcohol dehydrogenase - APC antigen-presenting cell - cpm counts per minute - E class II Mhc molecule onsisting of the E and A chains - Ir immune response - KLH key-hole limpet hemocyanin - Mhc major histocompatibility complex - PBS phosphate buffered salt solution - SD standard deviation - SI stimulation index - SN supernatant of Sendai-virus preparation     

Root and stem xylem embolism,stomatal conductance,and leaf turgor in Acer grandidentatum populations along a soil moisture gradient

The objective of this study was to determine how adjustment in stomatal conductance (g ^s) and turgor loss point (_tlp) between riparian (wet) and neighboring slope (dry) populations of Acer grandidentum Nutt. was associated with the susceptibility of root versus stem xylem to embolism. Over two summers of study (1993–1994), the slope site had substantially lower xylem pressures (_px) and g ^s than the riparian site, particularly during the drought year of 1994. The _tlp was also lower at the slope (-2.9±0.1 MPa; all errors 95% confidence limits) than at riparian sites (-1.9±0.2 MPa); but it did not drop in response to the 1994 drought. Stem xylem did not differ in vulnerability to embolism between sites. Although slope-site stems lost a greater percentage of hydraulic conductance to embolism than riparian stems during the 1994 drought (46±11% versus 27±3%), they still maintained a safety margin of at least 1.7 MPa between midday _px and the critical pressure triggering catastrophic xylem embolism (_pxCT). Root xylem was more susceptible to embolism than stem xylem, and there were significant differences between sites: riparian roots were completely cavitated at -1.75 MPa, compared with -2.75 MPa for slope roots. Vulnerability to embolism was related to pore sizes in intervessel pit membranes and bore no simple relationship to vessel diameter. Safety margins from _pxCT averaged less than 0.6 MPa in roots at both the riparian and slope sites. Minimal safety margins at the slope site during the drought of 1994 may have led to the almost complete closure of stomata (g _s=9±2 versus 79±15 mmol m^-2 s^-1 at riparian site) and made any further osmotic adjustment of _tlp non-adaptive. Embolism in roots was at least partially reversed after fall rains. Although catastrophic embolism in roots may limit the minimum for gas exchange, partial (and reversible) root embolism may be adaptive in limiting water use as soil water is exhausted.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat liver

Summary The substrate specificity and the intraperoxisomal localization of -hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37°C in a medium containing 3 mM CeCl₃, 100 mM NaN₃ and 5 mM of an -hydroxyacid in 0.1M of one of the following buffers: Pipes, Mops, Na-cacodylate,Tris-maleate, all adjusted to pH 7.8. Ten different -hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic andl--isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in theTris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections inTris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of -hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.Abbreviations -HAOX l-hydroxyacid oxidase - Argininic acid l--hydroxy--guanidinovaleric acid - Pipes piperazine-N,N-bis(2ethane sulfonic acid) - Mops 3(N-morpholino) propane sulfonic acid - Tris tris-(hydroxymethyl)-aminomethane - Luminol 5-amino-2,3 dihydrophthalazine-1,4-dione - GA glutaraldehyde     

Formation of secondary metabolism enzymes in the tylosin producerStreptomyces T59-235

Production of the macrolide antibiotic tylosin byStreptomyces T59-235 was inhibited in cultures containing high phosphate concentrations (30 mM P_i). Vegetative growth (dry weight increase, DNA and RNA synthesis) was hardly affected. Tylosin production began when macromolecule synthesis had slowed down to minimum level; in cultures with 30 mM P_i the onset of antibiotic production was retarded compared to cultures with low phosphate concentration (5 mM). The activities of three enzyme systems involved in tylosin biosynthesis (dTDP-D-glucose-4,6-dehydratase; dTDP-mycarose-forming enzyme system; SAM: macrocin-O-methyl transferase) were measured and found to be significantly lower in cultures with 30 mM P_i than in low phosphate cultures.Chloramphenicol, but not rifampicin, caused a rapid decrease of both tylosin formation rate and dTDP-D-glucose-4,6-dehydratase activity when added to tylosin producing cultures.Abbreviations P_i inorganic phosphate - dTDB 2-deoxythymidine diphosphate - SAM S-adenosyl-L-methionine - LP low phosphate (5 mM) - HP high phosphate (30 mM)     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe(III)-chelate reductase in roots of strategy I species

Stimulation of root Fe(III) reductase activity by iron additions to iron-deficient growth media may be the result of iron activation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase required for ethylene biosynthesis. Two different ethylene inhibitors, aminooxyacetic acid (AOA) (20 m; ACC synthase inhibitor) and cobalt (3 m CoCl₂; ACC oxidase inhibitor), were used to study the effects of iron supply and cobalt inhibition on ethylene action in controlling the activity of Fe(III)-chelate reductase in pea (Pisum sativum L.) roots. Supplying 20 gm m Fe(III)-N,N-ethylenebis[2-(2-hydroxypheyl)-glycine [Fe(III)-EDDHA] to either cobalt-treated, iron-deficient Sparkle (normal parent) or E107 (brz mutant genotype) pea seedlings reversed the negative effects of cobalt on root Fe(III)-reductase activity. Re-supplying 20 m Fe(III)-EDDHA to iron-deficient, AOA-treated seedlings did not enhance root Fe(III)-reductase. Apparently, cobalt competes with iron for the active site in ACC oxidase during ethylene synthesis. Inhibition of root reductase activity by cobalt treatment lowered manganese, zinc, magnesium and potassium content of mutant E107 pea seedlings. In contrast, iron enhancement of root reductase activity in iron-deficient, cobalt-treated E107 seedlings resulted in higher seedling accumulations of manganese, zinc, magnesium and potassium. These results support the hypothesis that root cell plasma membrane reductase activity plays a role in cation uptake by root cells.     

Cell wall anionic polymers and peptidoglycan of Actinoplanes philippinensis VKM Ac-647

The cell wall of Actinoplanes philippinesis VKM Ac-647 harbours several carbohydrate-containing anionic polymers. (1) The main polymer of the wall is of a poly(glycosylglycerol phosphate) nature. Its monomeric units — O--d-mannopyranosyl-(14)--d-galactopyranosyl-(11)-glycerol monophosphates — are connected by phosphodiester bonds involving the hydroxyl groups at glycerol C3 and galactose C6. There also are chains without mannosyl substitutents. The teichoic acid structure has been established by chemical analysis and with ¹H and ¹³C NMR spectroscopy. This is the first finding of a teichoic acid with mannosyl residues in a bacterial cell wall. (2) The phosphorylated mannan contains mannose and 2-O-methylmannose. Its core chain has -1,2; -1,3; and -1,6 substitutions as revealed by ¹³C NMR spectroscopy.The peptide unit of the peptidoglycan contains no l-alanine, instead of which position 1 is occupied by glycine; and diaminopimelic acid is represented, besides its meso- (or DD) form, by small amounts of its LL isomer.Abbreviations Gro glycerol - Gro2P glycerol-2 phosphate - APT attached-proton-test - P_tot total content of phosphorus - P_lab phosphorus mineralized in 7 min at 100°C - P_NA phosphorus of nucleic acids - P_stab stable phosphorus - T trace amounts