Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of γ to CT maximum (γ/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a ≥ 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.     

A highly divergedβ1 exon in theDR region of the human MHC: Sequence and evolutionary implications

TheDR subregion of the human major histocompatibility complex from aDR4 haplotype includes the well-characterizedDR _ga ,DR4 DR(MT3) andDR genes. In addition, the region between theDR and the proximalDR(MT3) genes contains several copies of conserved DR -related sequences. These repeated elements, numbered II, III, and IV, include the DR signal sequence and a region located further upstream. Further examination of these conserved sequences showed that DR first intron sequences are present at the 3 ends of these repeats. Progressively longer portions of the DR first intron are conserved from repeat II to repeat IV, producing a gradient of conservation. The most complete repeat element of repeats II, III, and IV is associated with a lone1 exon (DR ₁). Upon sequencing, (DR ₁). was found to contain several deleterious mutations, indicating that it is nonfunctional. (DR ₁). has accumulated a large number of replacement substitutions and mutations at positions which are invariant in1 domains from expressedDR genes: 77.8% of the nucleotide substitutions were replacement substitutions, and 41.5 % of the amino acids at invariant positions have been altered. Calculations based on these figures suggest thatDR 1 may have become inactive approximately 25 million years ago. There are, however, two histidine residues within a variable region which are unique toDR 1 and theDR4 gene, suggesting that they represent a gene pair which probably evolved by duplication of a singleDR chain gene.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-d) for culture initiation, and 5 M 2,4-d for subculturing, was optimal; 90% of 4 day-old Turbo seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-d, but supplemented with 2.22 M 6-benzylaminopurine and 0.27 M naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic Turbo calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using Nandu were within the same range.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Age and growth,reproduction and diet of a sublittoral population of the rock goby Gobius paganellus (Teleostei,Gobiidae)

Colonisation of extremely acidic waters (pH 3) by aquatic angiosperms occurs widely, but is poorly documented. Unlike acid rain affected and other naturally acidic aquatic ecosystems, waters with pH 3 usually have a high conductivity, with high concentrations of SO₄ ^2- and often high concentrations of Fe³⁺, other heavy metal ions and Al³⁺. Where Fe³⁺ concentration is high, as in many mine waters, it provides a strong buffering system. In such waters, the biogeochemical Fe cycle exerts over water chemistry and the availability of nutrients and carbon for organisms. Biological activity is limited by low concentrations of phosphorus and inorganic carbon (DIC), which in this pH range is essentially all in the form of dissolved CO₂. A number of angiosperms grow in such waters including Phragmites australis, Typha spp. and Juncus bulbosus, though the last is the only one reported to grow totally submerged in waters with pH 3 . J. bulbosus occurs in many lignite mining lakes in Lusatia (north eastern Germany) with pH 3. The characteristics and possible survival strategies for this and other species are discussed.     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli

Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of - and -subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K_m=0.77 M) than for 2-aminophenol (K_m=0.89 M) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the - and -subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the - and -subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Magnetic circular dichroism studies of myoglobin,hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q ₀₀- and Q_v-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q ₀₀-band to the A-term in the Q _v-band. The evidences are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the hemoglobin-like MCD, while the alkaline ferroperoxidase is characterized by the myoglobin-like MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoproteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-ion charge-transfer electronic transition which may be assigned as b() 3d. This charge-transfer band is strongly overlapped with usual B( - *) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Homologies in processing and sequence between the 23S ribosomal ribonucleic acids of Paracoccus denitrificans and Rhodopseudomonas sphaeroides

We have shown that mature 50S ribosomal subunits of Paracoccus denitrificans lack intact 23S rRNA, containing instead rRNAs of 0.56 (16S) and 0.37 (14S)x10⁶ molecular weight. Kinetic labelling studies showed these to be derived from a 1.02x10⁶ dalton precursor, which may itself derive from a larger and very transient 23S species. A similar pattern of rRNA processing has been previously described for Rhodopseudomonas sphaeroides, and we have compared, by Tl oligonucleotide catalog analysis, the smaller (14S) fragments of P. denitrificans and R. sphaeroides 23S rRNAs. These were shown to exhibit strong sequence homology, and comparisons of 14S-derived oligonucleotides to oligonucleotides from an in vitro-generated 13S fragment of Escherichia coli 23S rRNA suggest that P. denitrificans and R. sphaeroides 14S rRNAs arise from the 5-terminal portions of their respective 23S precursors. Results are considered to be consistent with the claim that P. denitrificans arose, by loss of photophosphorylation, from a member of the Rhodospirillaceae.Abbreviations E buffer 60 ml 2 M Tris base, 20 ml 3 M sodium acetate, 15 ml 0.2M disodium EDTA, 6 ml glacial acetic acid, 900 ml distilled water - HEPES N-2-hydroxymethyl piperazine-N-2-ethanesulfonic acid - TMK 5 mM Tris-Cl, 0.1 mM MgSO₄, 60 mM KCl. pH 7.3 - TM3 10 mM Tris-Cl, 1 mM MgCl₂, pH 7.3 - Tris tris (hydroxymethyl) aminomethane - SDS sodium dodecyl sulphate     

Evidence for carrier-mediated,energy-dependent uptake of urea in some bacteria

Evidence for the existence of an energy-dependent urea permease was found for Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5a1 by studying uptake of ¹⁴C-urea. Since intracellular urea was metabolized immediately, uptake did not result in formation of an urea pool. Evidence is based on observations that the in vivo urea uptake and in vitro urease activity differ significantly with respect to kinetic parameters, temperature optimum, pH optimum, response towards inhibitors and regulation. The K _m for urea uptake was 15–20 times lower (38 M and 13 M urea for A. eutrophus and K. pneumoniae, respectively) than the K _m of urease for urea (650 M and 280 M urea), the activity optimum for A. eutrophus was at pH 6.0 and 35°C for the uptake and pH 9.0 and 65°C for urease. Uptake but not urease activity in both organisms strongly decreased upon addition of inhibitors of energy metabolism, while in K. pneumoniae, potent inhibitors of urease (thiourea and hydroxyurea) did not affect the uptake process. Significant differences in the uptake rates were observed during growth with different nitrogen sources (ammonia, nitrate, urea) or in the absence of a nitrogen source; this suggested that a carrier is involved which is subject to nitrogen control. Some evidence for the presence of an energy-dependent uptake of urea was also obtained in Pseudomonas aeruginosa DSM 50071 and Providencia rettgeri DSM 1131, but not in Proteus vulgaris DSM 30118 and Bacillus pasteurii DSM 33.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenole     

The Effects of Hypoxia on Three Sympatric Shark Species: Physiological and Behavioral Responses

Behavioral and physiological responses to hypoxia were examined in three sympatric species of sharks: bonnethead shark Sphyrna tiburo, blacknose shark, Carcharhinus acronotus, and Florida smoothhound shark, Mustelus norrisi, using closed system respirometry. Sharks were exposed to normoxic and three levels of hypoxic conditions. Under normoxic conditions (5.5–6.4mg l^–1), shark routine swimming speed averaged 25.5 and 31.0cm s^–1 for obligate ram-ventilating S. tiburo and C. acronotus respectively, and 25.0cm s^–1 for buccal-ventilating M. norrisi. Routine oxygen consumption averaged about 234.6 mg O₂kg^–1h^–1 for S. tiburo, 437.2mg O₂kg^–1h^–1 for C. acronotus, and 161.4mg O₂ kg^–1 h^–1 for M. norrisi. For ram-ventilating sharks, mouth gape averaged 1.0cm whereas M. norrisi gillbeats averaged 56.0 beats min^–1. Swimming speeds, mouth gape, and oxygen consumption rate of S. tiburo and C. acronotus increased to a maximum of 37–39cm s^–1, 2.5–3.0cm and 496 and 599mg O₂ kg^–1 h^–1 under hypoxic conditions (2.5–3.4mg l^–1), respectively. M. norrisi decreased swimming speeds to 16cm s^–1 and oxygen consumption rate remained similar. Results support the hypothesis that obligate ram-ventilating sharks respond to hypoxia by increasing swimming speed and mouth gape while buccal-ventilating smoothhound sharks reduce activity.     

Characterization of solute/proton cotransport in plasma membrane vesicles from Ricinus cotyledons,and a comparison with other tissues

Plasma membrane vesicles, purified by aqueous two-phase partitioning, were used to investigate the presence of sugar and amino acid carriers in cotyledons and roots of Ricinus communis L. and in roots of red beet (Beta vulgaris L.). Artificial pH and electrical gradients were generated across the plasma membrane, and [¹⁴C]acetate and [¹⁴C]tetraphenylphosphonium were used to demonstrate the presence of an internal alkaline pH gradient and an internal negative membrane potential, respectively. In Ricinus cotyledons, uptake of sucrose was more strongly inhibited than that of glutamine by p-chloromercuribenzenesulphonic acid, phlorizin and phenylglyoxal. The sucrose transport system showed a high degree of substrate specificity with only the presence of maltose and phenyl--glucoside significantly affecting sucrose uptake; in contrast, the glutamine transport system was inhibited by a number of other amino acids. pH+gD-driven glutamine uptake showed saturation kinetics with a K _m of 0.35 mol · m^–3. Sucrose and glutamine -driven uptake was pH dependent with an optimum in the acidic range (pH 6.25) and a decrease at higher pH values. Vesicles obtained from cotyledons and roots of Ricinus showed different transport properties. In the cotyledons, gDH+gD-driven transport for both sucrose and glutamine were observed at similar levels; however, in the root tissue, pH--driven glutamine transport was the dominant uptake process. Uptake rates for glucose and fructose were low in the cotyledons whereas, in the roots, glucose and sucrose transport were slightly higher than that of fructose. In vesicles from red beet tissue there was a different uptake profile, with evidence of proton-coupled cotransport systems for sucrose and glucose, but lower uptake of glutamine and fructose. The results are discussed in relation to the reported different pathways for loading and unloading of solutes in these tissues.Abbreviations CCCP carbonyl cyanide-m-chlorophyenyl hydrazone - DEPC diethyl pyrocarbonate - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid - TPP tetraphenylphosphonium ion - gDH⁺ proton electrochemical potential gradient - membrane potential We would like to thank the SERC(UK) and the Royal Society for financial support.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate