Biphasic mineral nutrition of the submersed aquatic macrophyte Potamogeton pectinatus L.

The mineral nutrition of a clone of the submersed aquatic macrophyte Potamogeton pectinatus L. was examined in relation to the ability of the roots to mobilize N, P, K, S, Ca, Mg, dissolved inorganic C and micronutrients to the shoots from a constant small volume of sediment in the absence of one or more of these nutrients in the water phase. Survival, biomass production and shoot nutrient concentration values were measured after 35 days of growth under controlled conditions. Flower production and shoot morphology were also noted.The roots of P. pectinatus were capable of mobilizing sufficient P, N, S, K and micronutrients from the sediment to the shoots to meet normal growth requirements. In the absence of K from the water phase, Na replaced it, but the vigor of the plants suffered somewhat by the substitution. The roots were not capable of mobilizing sufficient Mg, Ca, or dissolved inorganic C from the sediment to the shoots to meet normal growth requirements. Survival and normal growth occurred with a minimum of 2 ppm Ca, 10 ppm Mg, and 0.5 meq HCO₃^? in the water phase. Water-phase Ca was necessary to prevent the toxicity of other cations such as Mg when present in the water phase.A seasonal periodicity in biomass production occurred under standardized environmental conditions, suggesting an internal regulation independent of obvious external signals.     

Trace metals in sea water. : C. S. Wong,Edward Boyle,Kenneth W. Bruland,J. D. Burton and Edward D. Goldberg. Nato Conference Series,Series IV: Marine Sciences,Volume 9, Plenum Press,New York and London,pp. xiv + 920, ISBN 0-306-41165-2, U.S. $ 115.00.

A comprehensive mechanism is proposed for the photolysis of transition metal complexes in equilibrium solutions. This involves the photo-oxidation, a possibility of energy transfer and several secondary thermal reactions, e.g. forward and back reactions with photochemically-formed H· radicals. The scheme includes the individual quantum yield (IQY) of each species. The photo-oxidations of FeSO₄, FeClO₄, FeCl₂ in aqueous acidic solutions were studied by continuous irradiation. The IQY of Fe_aq²⁺ (0.2), FeSo₄ (0.3), FeHSO₄⁺ (0.67), FeClO₄⁺ (0.06–0.2), FeCl⁺ (0.8), FeCl₂ (0.3) were determined. The IQY was found to be dependent on H⁺ concentration, due to competition with geminal recombination within the solvent cage. This dependence, and the efficiency of energy transfer between excited iron(III) species and iron(II) in ground state, are discussed.     

The effect of temperature on the self-association of S5 and on the association of S5 with S8 as determined by sedimentation equilibrium

Solutions of proteins S5 and S8 from the Escherichia coli 30 S ribosomal subunit have been examined by sedimentation equilibrium methods as a function of temperature for their behavior in solution as isolated components and in mixtures. The standard enthalpy and entropy at 4 °C for the isodesmic self-association of S5 were determined from a study over the temperature range of 3 to 33 °C to be 0.1 ± 0.9 kcal/mol and 18 ± 3 cal/(mol × deg), respectively. The protein S8 remained monomeric over the same range of temperature. The standard enthalpy and entropy at 4 °C for the association of S5 and S8 were determined on mixtures from a study over the temperature range of 3 to 27 °C to be ?0.4 ± 1.6 kcal/mol and 20 ± 6 cal/(mol × deg), respectively. Based on these values and the previously determined standard Gibbs free energies (S. H. Tindall and K. C. Aune, 1981, Biochemistry20, 4861–4866), the driving force for the self-association of S5 and the association of S5 with S8 could be interpreted as being derived from the expulsion of water upon ion pair formation at the interaction sites.     

Metal ions in biological processes. Volume 15. Zinc and its role in biology and nutrition. : Edited by Helmut Sigel. (Institute of Inorganic Chemistry,University of Basel.) Marcel Dekker,Inc., New York,N.Y. and Basle,Switzerland, 1983. 520 pp., bound,illustrated. SFr. 198.

Seven new complexes where the chelate ring was nucleophilically substituted were synthesized by the reactions of asymmetrical tetradentate Schiff base complexes of nickel(II) and copper(II) with benzenethiols and thiolycollic ehtyl ester. They were characterized by means of elemental analyses, electronic and ¹H-NMR spectra. Kinetics of the nucleophilic ring-substitution reactions were spectrophotometrically studied in a benzene solution. A four-centered intermediate is proposed for the reactions.     

Studies on the metal—amide bond. XVII. The crystal and molecular structure of the α-form of aqua[N,N′-bis(2′-pyridinecarboxamido)-1,3-propane] copper(II) dihydrate

α-Aqua[N,N′-bis(2′-pyridinecarboxamido)-1,3-propane]copper(II) dihydrate, C₁₅H₂₀N₄O₅Cu, is monoclinic, space group P2₁/c, with a = 11.719(2), b = 13.092(2), c = 12.663(2) Å, β = 119.56(1)°, Z = 4. The structure was refined to R = 0.026 for 2398 diffractometer data using full-matrix least-squares methods. The copper atom is five-coordinate with the N₄-tetradentate ligand encompassing the base of a distorted square-based pyramid which is appreciably distorted towards a trigonal bipyramid [average Cu-N(amide) 1.950(2), Cu-N(pyridine) 2.043(2) Å, N(amide)-Cu-N(amide) 94.5(1), N(pyridine)-Cu-N(pyridine) 100.2(1)°] and with the copper atom lying 0.27 Å above the N₄ plane towards the apical water molecule [Cu-O 2.236(2) Å]. The central six-membered chelate ring adopts a skewed boat conformation and the enforced strain in the molecule results in non-planar distortions in the pyridine rings with only small distortions in the amide groups. The molecules pack in sheets parallel to (10$\text{1}$) and the hydrogen-bonding network involves the water molecules and the amide oxygen atoms of the ligand.     

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.     

Catalytic conformation of carboxypeptidase A. The structure of a true reaction intermediate stabilized at subzero temperatures

The structure of the mixed anhydride, acyl-enzyme intermediate of the esterolytic reaction of carboxypeptidase A is characterized by application of cryoenzymologic, magnetic resonance, and molecular graphics methods with use of the Co²⁺-substituted enzyme and the specific spin-label ester substrate O-3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-2-oyl-l-β-phenyllactate. A radial separation of 7·7 Å between the active site Co²⁺ and the nitroxide group in the low temperature-stabilized acyl-enzyme intermediate is determined on the basis of their spin-spin (dipole-dipole) interactions. Application of molecular graphics techniques shows that the only configuration of the substrate that is sterically accommodated by the active site yields a calculated metal ion-to-nitroxide distance of 7·8 Å. Steric accommodation of the spin-label in the active site requires severe torsional distortion around the aliphatic double bond of the propenoyl side-chain. Examination of the structure of the enzyme: spin-label intermediate reveals that the distortion arises from steric interactions of the pyrrolinyl group with the protein at a position that corresponds to the site occupied by the penultimate amide residue of an oligopeptide substrate from the site of cleavage. Together with kinetic data showing that hydrolysis of the spin-label is governed by rate-limiting deacylation, the results indicate that geometric distortion of substrates by secondary interactions with the enzyme, in general, is an obligatory part of the catalytic action of carboxypeptidase A. When viewed with respect to requirements for stereoelectronic control of bond cleavage in tetrahedral adducts of esters and amides (Deslongchamps, 1975) the results suggest that torsional distortion during catalysis results in rotation around the scissile bond of the substrate, and that this rotation is required to form the mixed anhydride reaction intermediate. These findings further support the interpretation that the hydrolysis of esters and amides catalyzed by carboxypeptidase A proceeds according to similar mechanisms except that formation of the mixed anhydride is rate-determining in peptide hydrolysis while deacylation of the mixed anhydride is rate-limiting in ester hydrolysis.Additionally, in this study application of the extension of the theory of the Solomon-Bloembergen-Morgan equations derived by Lindner (1965) for paramagnetic metal ions with S ≥ 1 demonstrates that the zero-field splitting of the high-spin Co²⁺ in the metal-substituted enzyme has no significant influence in determination of the relaxation enhancement of solvent protons by the active site metal ion.     

Metal ions in biological systems. vol. XVIII. Circulation of metals in the environment : Edited by Helmut Sigel,Marcel Dekker,New York and Basle, 1984, 397 pp.

The solvent, pressure and temperature dependencies of the lowest energy metal to ligand charge transfer absorption bands were studied for a series of complexes of the type Mo(CO)₄(N$\text{N}$), where N$\text{N}$ = 2,2′-bipyridine, 1,10-phenanthroline and biacetylbis(phenylimine). Throughout the series of complexes the absorption bands shift to shorter wavelength in more polar solvents or on increasing the pressure in a particular solvent, but to longer wavelengths on increasing temperature. These main tendencies can be accounted for in terms of solvent polarity and its dependence on pressure and temperature.     

The synthesis of 8-(6-aminohexyl)-amino-GMP and its applications as a general ligand in affinity chromatography.

The steady-state kinetic behaviors of the five rabbit adrenal norepinephrine N-methyl transferase isozymes have been compared with particular reference to substrate inhibition patterns. Four distinct substrate inhibition patterns were observed. The E-1 isozyme was not subject to inhibition by either substrate, while the E-2 isozyme was inhibited by both substrates. The E-3 and E-4 isozymes were inhibited by norepinephrine only, while E-5 is inhibited only by S-adenosylmethionine. The substrate inhibition constants were sufficiently small in relation to the Michaelis constants to make substrate inhibition an important factor in regulation of activities of the isozymes.     

Genes and pseudogenes for human U2 RNA: Implications for the mechanism of pseudogene formation

Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3′ end. The conserved 3′-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus.The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5′ and 3′ sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5′ side the homology continues beyond the Alu repeats whereas on the 3′ side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.     

A model chromatin assembly system. Factors affecting nucleosome spacing

Poly[d(A-T)].poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 degrees C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.     

Biosynthesis of monoterpenes: hydrolysis of bornyl pyrophosphate, an essential step in camphor biosynthesis, and hydrolysis of geranyl pyrophosphate, the acyclic precursor of camphor, by enzymes from sage (Salvia officinalis).

Soluble enzyme preparations from sage (Salvia officinalis) leaves catalyze the hydrolysis of (+)-bornyl pyrophosphate to (+)-borneol, which is an essential step in the biosynthesis of the cyclic monoterpene (+)-camphor [(1R,4R)-bornan-2-one] in this tissue. Chromatography of the preparation on Sephadex G-150 allowed the separation of two regions of bornyl pyrophosphate hydrolase activity. One region was further separated into a pyrophosphate hydrolase and a monophosphate hydrolase by chromatography on hydroxylapatite, but the other contained pyrophosphate and monophosphate hydrolase activities which were inseparable by this or any other chromatographic technique tested. Each phosphatase and pyrophosphatase activity was characterized with respect to molecular weight, pH optimum, response to inhibitors, K_m for bornyl phosphate or bornyl pyrophosphate, and substrate specificity, and each activity was distinctly different with regard to these properties. One pyrophosphatase activity was specific for pyrophosphate esters of sterically hindered monoterpenols such as bornyl pyrophosphate. The other preferred pyrophosphate esters of primary allylic alcohols such as geranyl pyrophosphate and neryl pyrophosphate, which are precursors of cyclic monoterpenes, and it hydrolyzed geranyl pyrophosphate at faster rates than neryl pyrophosphate. The monophosphate hydrolase activities were similar in substrate specificity, showing a preference for phosphate esters of primary allylic alcohols. The terpenyl pyrophosphate hydrolase exhibiting specificity for bornyl pyrophosphate may be involved in camphor biosynthesis in vivo, while the terpenyl pyrophosphate hydrolase more specific for geranyl pyrophosphate was shown to be a source of potential interference in studies on monoterpene cyclization processes.     

S′2-P′2 interaction and the trypsin anilide hydrolysis

Both an increase in the bulkiness of the leaving group and the presence of aliphatic alcohols produce a rate enhancement in the trypsin hydrolysis of acetyl-l-Lys p-acyl anilides. The effects are not due to the electronic nature of p substituents and the better the substrate, the lower the degree of activation by aliphatic alcohols. These favorable steric effects are interpreted as providing evidence for the location of the secondary binding site and its operation as regulatory site in the hydrolysis of low molecular substrates by trypsin.     

Synthesis and accumulation of an extremely stable high-energy phosphate compound by muscle, heart, and brain of animals fed the creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine)

A new creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine), has been synthesized and compared with other synthetic analogs of creatine as a substrate for creatine kinase under both in vitro and in vivo conditions. Reactivity with rabbit muscle creatine kinase at 2 mM and pH 7.0 occurred in the order: creatine greater than cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) greater than N-ethylguanidinoacetate greater than N-propylguanidinoacetate greater than guanidinoacetate greater than N-methyl-3-guanidinopropionate greater than 3-guanidinopropionate greater than homocyclocreatine. Homocyclocreatine was 10,000-fold less active than creatine. In the reverse direction at 0.2 mM and pH 7.0: creatine-P greater than N-ethylguanidinoacetate-P greater than cyclocreatine-P much greater than homocyclocreatine-P. Homocyclocreatine-P was 200,000-fold less active than creatine-P. The phosphoryl group transfer potential of homocyclocreatine-P was estimated to be 2 kcal/mol lower than that of creatine-P. Chicks fed 5% homocyclocreatine for 16 days synthesized and accumulated homocyclocreatine-P in breast muscle (32 mumol/g wet wt), leg muscle (24 mumol/g), heart (7 mumol/g), intestine (8.5 mumol/g), and brain (2.4 mumol/g). During ischemia homocyclocreatine-P was utilized by muscle much more slowly for the regeneration of ATP than was creatine-P or cyclocreatine-P. Our results suggest that in tissues of homocyclocreatine-fed animals subjected to a sudden large increase in work load or to ischemia, the residual creatine-P system would rapidly equilibrate with the adenylate system at the new lower cytosolic phosphorylation potential, whereas in the same cytosol the (homocyclocreatine-P)/(homocyclocreatine) ratio would exhibit a hysteresis or memory effect and reflect for a considerable period of time the earlier higher (ATP)/(free ADP) ratio rather than the actual lower (ATP)/(free ADP) ratio.     

Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology

Group-directed hydrophobic modification of membrane-integrated protein segments by arylisothiocyanates is applied to bacteriorhodopsin. Labeling of purple membrane with phenylisothiocyanate and 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate results in covalent modification of a unique lysine epsilon-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothiocyanate binding site by established sequencing techniques. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Furthermore, the acid-induced bathochromic shift of the bound chromophore reagent is not observed following acidification of 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-labeled purple membrane. The modification thus occurs in the hydrophobic membrane domain, providing further evidence for intramembraneous disposition of the modified protein segment. Light-induced proton translocation is preserved in reconstituted vesicles containing either phenylisothiocyanate-modified or 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-modified bacteriorhodopsin.     

Complexes of vitamin B6. XVI. Kinetics and reaction mechanism of iron(III) complexes with pyridoxal-5′-phosphate

The stopped flow technique has been used to study the kinetics of complex formation of iron(III) with pyridoxal-5-phosphate (PLP) in the pH range 1.00–2.50, and in the temperature range 18 °C– 30 °C, at an ionic strength of. 0.50 M (NaCl). From the initial concentration dependence of PLP (T_PLP,) of the reaction rate it can be shown that two kinetic steps can be represented as: k_obs′ = m_i + m_i′_PLP where m_i and m_i′ are pH-dependent parameters. The calculated activation data are δE* = 23.2 ± 1.8 kcal mol^?1 and 10.98 ± 0.53 kcal mol^?1 for the first and second kinetic steps, respectively and δS* are ?20.50 ± 5.96 e.u. and 24.62 ± 1.81 e.u., respeetively.