Synthesis of plastoquinone-9 and phytylplastoquinone from homogentisate in lettuce chloroplasts

Chloroform-soluble extracts of unpurified chloroplast preparations of lettuce, pea and spinach and of class I lettuce chloroplasts that have been incubated in the light with [methylene-³H]homogentisate contain ³H-labelled plastoquinones-9 and -8 (minor homologue), 2-demethylplastoquinones-9 and -8 (minor homologue), pytylplastoquinone and 2-demethylphytylplastoquinone.. The absence of demethylquinols, the presumed precursors of the dimethylquinones, from the extracts to the fact that no precautions were taken in the extraction procedure to present their oxidation to the corresponding quinones.In unpurified lettuce chloroplasts the synthesis of these compounds from [methylene-³H]homogentisate is Mg²⁺-dependent and it is stimulated by light. The addition of isopentenyl pyrophosphate to the incubation mixtures increases the amounts of both groups of quinones (polyprenyl quinones and phytyl quinones) synthesised in the light and the amounts of polyprenyl quinones synthesised in the dark. Replacement of isopentenyl pyrophosphate with a source of preformed polyrenyl pyrophosphates brings about a marked rise in the amounts of polyprenyl quinones synthesized. This rise in polyprenyl quinone synthesis is further increased if the chloroplats are subjects to osmotic shock. The presence of S-adenosylmethionine increases the amounts of dimethylquinones synthesized at the expense of the demethylquinones. The implied precursor-product relationships between 2-demethylphytylplastoquinone (quinol?) and phytylplastoquinone and between the 2-demethylplastoquinones (quinols) and plastoquinones were verified in a pulse-labelling experiment. Confirmation that these quinones, or their corresponding quinols, are synthesized.     

Characterization of undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum

A soluble long-chain polyprenyl pyrophosphate synthetase has been isolated from Lactobacillus plantarum and partially purified by DEAE-cellulose chromotography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate and Δ³-isopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with C₅₅-isoprenyl pyrophosphate phosphatase (Micrococcus lysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker's yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited R_fs which corresponded to those of authentic undecaprenol isolated from Lactobacillus plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.     

Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves.

Pineapple leaves contain a pyrophosphate-dependent 6-phosphofructokinase which has been partially purified and characterized. In crude extracts the pyrophosphate-dependent activity is 10 to 20-fold higher than the ATP-dependent activity. The partially purified activity is near 2.5 μmol Fru-1,6-P₂ formed/min/mg protein. In the reaction 1 Fru-1,6-P₂ is formed per 1 pyrophosphate consumed. The enzyme exhibits a pH optimum of 8.0 and the activity is stimulated by Mg⁺⁺. The discovery of a pyrophosphate-dependent 6-phosphofructokinase in pineapple leaves indicates pyrophosphate can serve as an energy source for synthetic reactions in pineapple and perhaps in other plants as well.     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament

Actin filament bundles isolated from Limulus sperm were used for quantitative electron microscope studies of F-actin assembly. The assembly rate constants were calculated. In addition, the critical concentrations (C_os) for both filament ends were directly determined. In 75 mM KCI and 1–5 mM Mg⁺⁺, the C_os were 0.1 μM and 0.5 μM for the barbed and pointed ends, respectively. Substitution of Ca⁺⁺ (20–200 μM) for Mg⁺⁺ resulted in C_os of 0.4 μM for both filament ends. Consistent with these findings, filament growth occurred only from the barbed ends of Limulus bundles “seeded” into F-actin solutions in KCI and Mg⁺⁺. Finally, filaments originally grown from the pointed filament ends of Limulus bundles were gradually lost as the actin solution reached steady state. These results demonstrate that actin filaments can “treadmill” under physiological conditions, albeit at very slow rates.     

Lactobacillus plantarum undecaprenyl pyrophosphate synthetase: purification and reaction requirements.

Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ³-[1-¹⁴C]isopentenyl pyrophosphate and Δ³-2R-[2-³H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ exhibiting comparable activities.     

Reversible inhibition of skeletal muscle phosphoprotein phosphatase by ATP, phosphate and fluoride.

A phosphoprotein phosphatase preparation which showed activity towards glycogen synthase, phosphorylase, phosphorylase kinase, and phosphohistones was reversibly inhibited (70–90%) by preincubation with free ATP (apparent K_i about 0.3 mM). Other nucleotides (ADP2 (apparent K_a 3μM) prior to assay. Other divalent metals (Co⁺⁺ > Zn⁺⁺ > Mg⁺⁺) were partially effective in reversing the inhibition. It is concluded that ATP by virtue of its special structure and metal binding capacity possibly removes a catalytically important metal ion from the enzyme.     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Activation of tubulinyl-tyrosine carboxypeptidase by spermine,spermidine and putrescine

Spermine, spermidine and putrescine produce dose dependent stimulation of the invitro tubulinyl-tyrosine carboxypeptidase. Maximal stimulation was obtained with spermine, spermidine or putrescine at 0.06 mM, 1 mM and 6 mM, respectively. At higher concentrations, the enzyme activity was inhibited. The enzyme was also activated by Mg⁺⁺; the concentration formaximal effect was 4–6 mM. The stimulation produced by optimal concentration of each amine was unaffected by Mg⁺⁺ up to 2 mM; higher concentration of Mg⁺⁺ showed inhibitory effect. At optimal Mg⁺⁺ concentration, the carboxypeptidase activity was inhibited by increasing amine concentration. The amines at 0.5 or 5 mM did not produce any effect on the incorporation of tyrosine catalyzed by tubulin tyrosine ligase.     

Polyprenyl pyrophosphate–p-hydroxybenzoate polyprenyltransferase activity in mitochondria of broad-bean seeds and yeast (Short Communication)

Cell-free homogenates prepared from broad-bean seeds and yeast cells are capable of synthesizing 4-carboxy-2-polyprenylphenols from p-hydroxybenzoate and either isopentenyl pyrophosphate or protein-bound polyprenyl pyrophosphates (produced by incubating a Micrococcus lysodeikticus extract with isopentenyl pyrophosphate). The mitochondria contained all the polyprenyl pyrophosphate-p-hydroxybenzoate polyprenyltransferase activity; however, unlike the homogenates they could not synthesize a side chain from isopentenyl pyrophosphate and had to be provided with protein-bound polyprenyl pyrophosphates.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Biosynthesis of monoterpenes: preliminary characterization of bornyl pyrophosphate synthetase from sage (Salvia officinalis) and demonstration that Geranyl pyrophosphate is the preferred substrate for cyclization.

Previous studies with soluble enzyme preparations from sage (Salvia officinalis) demonstrated that the monoterpene ketone (+)-camphor was synthesized by the cyclization of neryl pyrophosphate to (+)-bornyl pyrophosphate followed by hydrolysis of this unusual intermediate to (+)-borneol and then oxidation of the alcohol to camphor (R. Croteau, and F. Karp, 1977, Arch. Biochem. Biophys.184, 77–86). Preliminary investigation of the (+)-bornyl pyrophosphate synthetase in crude preparations indicated that both neryl pyrophosphate and geranyl pyrophosphate could be cyclized to (+)-bornyl pyrophosphate, but the presence of high levels of phosphatases in the extract prevented an accurate assessment of substrate specificity. The competing phosphatases were removed by combination of gel filtration on Sephadex G-150, chromatography on hydroxylapatite, and chromatography on O-(diethylaminoethyl)-cellulose. In these fractionation steps, activities for the cyclization of neryl pyrophosphate and geranyl pyrophosphate to bornyl pyrophosphate were coincident, and on the removal of competing phosphatases, the synthetase was shown to prefer geranyl pyrophosphate as substrate ($\text{V}\text{K}{}_{\mathrm{m}}$ for geranyl pyrophosphate was 20-fold that of neryl pyrophosphate). No interconversion of geranyl and neryl pyrophosphates was detected. The partially purified bornyl pyrophosphate synthetase had an apparent molecular weight of 95,000, and required Mg²⁺ for catalytic activity (K_m for Mg²⁺ ~ 3.5 mm). Mn²⁺ and other divalent cations were ineffective in promoting the formation of bornyl pyrophosphate. The enzyme exhibited a pH optimum at 6.2 and was strongly inhibited by both p-hydroxymercuribenzoate and diisopropylfluorophosphate. Bornyl pyrophosphate synthetase is the first monoterpene synthetase to be isolated free from competing phosphatases, and the first to show a strong preference for geranyl pyrophosphate as substrate. A mechanism for the cyclization of geranyl pyrophosphate to bornyl pyrophosphate is proposed.     

Substrate specificity of cis-prenyltransferase in rat liver microsomes.

Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with Km values of 24 and 5 microM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent Km of 36 microM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyl 12-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.     

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase in mung beans and its activation by D-fructose 1,6-bisphosphate and D-glucose 1, 6-bisphosphate

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase was detected in extracts of mung bean sprouts, the first such detection in C₃ plants. The enzyme had an absolute requirement for a divalent metal (Mg⁺⁺) as well as for D-fructose 6-phosphate and inorganic pyrophosphate. An examination of anomalous kinetics revealed that the enzyme was activated by a product of the reaction, D-fructose 1,6-bisphosphate; micromolar concentrations of this effector increased the activity of the enzyme about 20-fold. D-Glucose 1,6-bisphosphate at higher concentrations could substitute for D-fructose 1,6-bisphosphate as an activator, but not as a substrate in the reverse reaction. The enzyme was fully active under conditions wherein ATP: D-fructose-6-phosphate 1-phosphotransferase from the same source was inhibited >99% (e.g., in the presence of 10 μM phosphoenolpyruvate).     

Protein synthesis in rabbit reticulocytes XXII+: a heat stable dialyzable factor (EIF-I*) modulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA_f·EIF-1·GTP in the absence of Mg⁺⁺ and the preformed complex is stable to Mg⁺⁺. However, with homogeneous preparations of EIF-1, addition of Mg⁺⁺ during the initial formation of the ternary complex strongly inhibits the complex formation.A heat stable dialyzable factor (EIF-1¹) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-1¹ restores the Met-tRNA_f binding activity of EIF-1 in the presence of 1 mM Mg⁺⁺; in the presence of EIF-1¹, Met-tRNA_f binding by EIF-1 shows a sharp Mg⁺⁺ optimum around 1 mM. EIF-1¹ is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-1¹ preparation also strongly inhibits Met-tRNA_f binding to EIF-1 in the absence of Mg⁺⁺ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Ion metabolism in a halophilic blue-green alga,Aphanothece halophytica

The intracellular ion content of the halophilic blue-green alga, Aphanothece halophytica was studied as a function of age, external sodium and external potassium concentration. Intracellular Na⁺ was found to be about 0.38 millimoles/g dry mass. Intracellular K⁺ concentrations were as high as 1 M and varied directly with external salinity. Intracellular Ca⁺⁺ and Mg⁺⁺ were in the range previously reported for fresh water blue-green algae despite their extremely high extracellular concentrations. Average cell size is consistent at room temperature with two exceptions. When the outside K⁺ is lower than 6.5 mM the cells tend to be smaller with less intracellular K⁺ and high Ca⁺⁺. In stationary phase cultures the cells are larger with high intracellular Mg⁺⁺ and low K⁺.