The substrate specificity, tissue specificity and regulation of the 5' deiodination systems in rat liver and kidney tissues

Studies were carried out to compare the 5' deiodination reactions of thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) in rat liver and kidney homogenates. The 5'-deiodinase activity was assayed by the 3, 5, 3'-triiodothyronine (T3) produced from T4 or by the 125I-iodide released from 125I-rT3. The two 5' deiodination reactions had similar ranges of optimal pH, incubation temperature, and apparent Km, T4 1.1 and rT3 1.3 microM. However, the apparent Vmax values for T4 and rT3 deiodination reactions were 0.9 and 220 pmol/mg protein/min, respectively. Both reactions were stimulated by thiol reagent but only rT3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil (PTU) on the 5' deiodination of rT3 was 50 times as great as that of T4. Only the 5' deiodination of rT3 was inhibited by low concentrations of calcium and magnesium. The 5' deiodination reactions in the liver and kidney tissues showed very similar substrate specificity. However, only the hepatic deiodinase activity was reduced to 60-65% of the control value after fasting, whereas the renal 5'-deiodinase activity was unaffected or even enhanced by fasting up to 72 hours. The results showed the existence of a diverse and complex 5' deiodination system in the rat tissues which is comprised of multiple similar but distinct 5'-deiodinase enzymes with respect to their substrate specificity, tissue specificity and regulation.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

Theoretical study of enzymatically catalyzed tautomerization of carbon acids in aqueous solution: quantum calculations and steered molecular dynamics simulations

The thermodynamics and kinetics of enzymatically assisted reactions of carbon acids were studied theoretically in this work. Quantum electronic (QE) structure calculations and steered molecular dynamics (SMD) simulations were carried out. Three 3-butenal tautomerization reactions that proceed from the β,γ-unsaturated reactant (R) to the α,β-unsaturated carbon acid product (P) and occur in two elementary steps through an intermediate (I) were studied, ignoring or including the surrounding aqueous medium in the calculations. The Gibbs free energies of activation of the R ? I enolization and I ? P ketonization steps were found to decrease considerably when residues simulating enzymes were introduced into these processes. Although the processes became slightly more favorable thermodynamically when the solution was included in the simulations, they became less favorable kinetically. The results from SMD simulations of these reactions were qualitatively consistent with the values we obtained using QE as well as those found by other authors in similar studies. Our simulations also allowed us to perform a detailed study of these reactions in solution.     

Investigation of the selectivity of hydrogen abstraction in the nonenzymatic formation of hydroxyeicosatetraenoic acids and leukotrienes by autoxidation

The biosynthetic conversions of arachidonic acid to hydroperoxyeicosatetraenoic acids (HPETEs) and the further conversion of leukotriene epoxides are accompanied by stereoselective hydrogen abstraction from the reaction substrate. Furthermore, this hydrogen removal has always been found to occur in fixed stereochemical relationship to carbon-oxygen chiral center(s) in the substrate or product. We have used stereospecifically labeled 10-3H-substrates with 14C internal standard to investigate whether the same relationships bear in HPETE and leukotriene formation during autoxidation. After autoxidation of labeled arachidonate, both the 8(R)- and 8(S)-HPETE enantiomers (resolved as diastereomer derivatives) and the 12(RS)-HPETE were observed to retain 41-47% 3H relative to the starting material. In autoxidative formation of leukotrienes from labeled 15(S)-HPETE the four main leukotrienes, including two derived from 14,15-leukotriene A4 hydrolysis, were observed to have retained an average of 45% 3H. Primary and secondary isotope effects were found to accompany these reactions. The results prove that stereorandom hydrogen abstraction occurs in autoxidation and that the hydrogen loss bears no stereochemical relationship to chiral oxygen center(s) in the HPETE product, (8(R) or 8(S], or the 15(S)-hydroperoxy substrate. We conclude that the chiral features of the biosynthetic reactions are a reflection of enzymatic control of stereochemistry. Nonetheless, the findings of primary and secondary isotope effects in autoxidation which are similar to those observed in the analogous biosynthetic reactions suggests that, except for stereochemical control, the autoxidative and enzymatic reactions may be mechanistically similar.     

Quantitative Comparison of Catalytic Mechanisms and Overall Reactions in Convergently Evolved Enzymes: Implications for Classification of Enzyme Function

Functionally analogous enzymes are those that catalyze similar reactions on similar substrates but do not share common ancestry, providing a window on the different structural strategies nature has used to evolve required catalysts. Identification and use of this information to improve reaction classification and computational annotation of enzymes newly discovered in the genome projects would benefit from systematic determination of reaction similarities. Here, we quantified similarity in bond changes for overall reactions and catalytic mechanisms for 95 pairs of functionally analogous enzymes (non-homologous enzymes with identical first three numbers of their EC codes) from the MACiE database. Similarity of overall reactions was computed by comparing the sets of bond changes in the transformations from substrates to products. For similarity of mechanisms, sets of bond changes occurring in each mechanistic step were compared; these similarities were then used to guide global and local alignments of mechanistic steps. Using this metric, only 44% of pairs of functionally analogous enzymes in the dataset had significantly similar overall reactions. For these enzymes, convergence to the same mechanism occurred in 33% of cases, with most pairs having at least one identical mechanistic step. Using our metric, overall reaction similarity serves as an upper bound for mechanistic similarity in functional analogs. For example, the four carbon-oxygen lyases acting on phosphates (EC 4.2.3) show neither significant overall reaction similarity nor significant mechanistic similarity. By contrast, the three carboxylic-ester hydrolases (EC 3.1.1) catalyze overall reactions with identical bond changes and have converged to almost identical mechanisms. The large proportion of enzyme pairs that do not show significant overall reaction similarity (56%) suggests that at least for the functionally analogous enzymes studied here, more stringent criteria could be used to refine definitions of EC sub-subclasses for improved discrimination in their classification of enzyme reactions. The results also indicate that mechanistic convergence of reaction steps is widespread, suggesting that quantitative measurement of mechanistic similarity can inform approaches for functional annotation.     

Synthesis and antioxidant activity of new lipophilic dihydropyridines

Dihydropyridines (DHPs) obtained from Hantzsch multicomponent reactions are an important pharmaceutical class of compounds marketed as antihypertensive (e.g., nifedipine, nitrendipine, and amlodipine) drugs. This study synthesized new symmetrical and unsymmetrical long-chain fatty DHPs using multicomponent reactions under metal-free conditions with sulfamic acid as a catalyst. The DHPs were tested for antioxidant activity using three different methods. The insertion of a long chain into the DHP core contributed to antioxidant potential, and compounds derived from nitro aldehydes have better antioxidant potential than the antihypertensive drug nifedipine. In addition, fatty analogs to nifedipine derived from palmitic and oleic chains showed similar antioxidant activity to the common standards butylated hydroxytoluene and vitamin E. These results showed that our new synthesized products may find novel applications as antioxidant additives or for tools for use in drug discovery.     

Histochemical reactions of gastrointestinal mucosubstances with orcein, high iron diamine and Alcian blue after prior oxidation of tissue sections.

The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.     

Vitamin E analogue Trolox C. E.s.r. and pulse-radiolysis studies of free-radical reactions.

The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were examined by using the pulse-radiolysis technique. The results demonstrate that Trolox C may undergo rapid one-electron-transfer reactions as well as hydrogen-transfer processes; the resulting phenoxyl radical is shown to be relatively stable, in common with the phenoxyl radical derived from vitamin E. The reactions between the Trolox C phenoxyl radical and a variety of biologically relevant reducing compounds were examined by using both pulse radiolysis and e.s.r. The results demonstrate that the Trolox C phenoxyl radical is readily repaired by ascorbate (k = 8.3 x 10(6) dm3.mol-1.s-1) and certain thiols (k less than 10(5) dm3.mol-1.s-1) but not by urate, NADH or propyl gallate. Evidence from e.s.r. studies indicates that thiol-containing compounds may also enter into similar repair reactions with the alpha-tocopherol phenoxyl radical. Kinetic evidence is presented that suggests that Trolox C may 'repair' proteins that have been oxidized by free radicals.     

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.     

Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit

Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate to yield propionyl-CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer of beta(8)alpha(8) barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S-based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites.     

Multiple Group-specific Antigen Components of Avian Tumor Viruses Detected with Chicken and Hamster Sera

Multiple group-specific (gs) components of the avian leukosis-sarcoma viruses were detected by immunodiffusion (Ouchterlony) tests with sera from hamsters bearing tumors induced by sarcoma viruses and with sera from adult chickens immunized with avian sarcoma or leukosis viruses. Immune hamster sera detected up to four components, whereas chicken sera detected at least one. The hamster and chicken sera identified a similar antigen, as indicated by reactions of identity. Relatively few chicken sera containing neutralizing antibody to avian sarcoma or leukosis viruses reacted in immunodiffusion with the gs antigen. The gs components were released from the virion by various means of disruption, including freezing and thawing. Tests with tissues from normal chickens and from chickens with Marek's disease failed to demonstrate any reactions with hamster or chicken gs antiserum.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections

Summary High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-HID and ox-HID) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive mucosubstances were found in the foveolar epithelium of the stomach and in goblet cells of small and large bowel.The study was supported by grants from Sigrid Juselius Foundation and Paulo Foundation, Helsinki, Finland     

Hydroformylation of alkenes and alkynes using a heterobinuclear Rh---W catalyst

Hydroformylation reactions of a series of alkenes and alkynes have been carried out using the heteronuclear Rh---W catalyst, (CO)₄ hH(CO)(PPh₃) (1). The results of these reactions have been compared with corresponding reactions using [Rh(OAc)₂]₂ as catalyst. Catalysis of a reaction of styrene using 1 gave a very high yield of the branched chain aldehyde containing only a trace of the straight chain isomer. Reactions of the phosphinoalkene, Ph₂P(CH₂)₃CH=CH₂ (7) and the corresponding alkyne, Ph₂P(CH₂)₃CCH (11) gave similar products using either catalyst system with the alkryne reaction being significantly slower. Reaction of the alkenyl dithiane, H---CH₂CH=CH₂ (2), using the Rh---W catalyst (1) gave a higher ratio of linear to branched aldehydes (47 linear:53 branched) than the corresponding reaction using [Rh(OAc)₂]₂ (25 linear:75 branched). Reactions of vinyl acetate using 1 as catalyst gave a significant amount of linear aldehyde in contrast to reactions using [Rh(OAc)₂]₂ but reactions of allyl acetate gave similar products for both catalyst systems.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections.

High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-Hid and ox-Hid) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive and in goblet cells of small and large bowel.     

Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride

Rhizopus niveus glucoamylase and Arthrobacter globiformis glucodextranase, which catalyze the hydrolysis of starch and dextrans, respectively, to form D-glucose of inverted (beta) configuration, were found to convert both alpha- and beta-D-glucosyl fluoride to beta-D-glucose and hydrogen fluoride. Each enzyme directly hydrolyzes alpha-D-glucosyl fluoride but utilizes th beta-anomer in reactions that require 2 molecules of substrate and yield glucosyl transfer products which are then rapidly hydrolyzed to form beta-D-glucose. Various D-glucopyranosyl compounds serve as acceptors for such reactions. Mixtures of beta-D-glucosyl fluoride and methyl-alpha-D-glucopyranoside[14C], incubated with either enzyme, yielded both methyl-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-[14C]glucopyranoside and methyl-alpha-D-glucopyranosyl-(1 leads to 6)-alpha-D-[14C]glucopyranoside. Glucoamylase produced more of the alpha-maltoside; glucodextranase produced more of the alpha-isomaltoside. Thus, both "exo-alpha-glucan hydrolases" emerge as glucosylases that catalyze stereospecifically complementary hydrolytic and transglucosylative reactions with glucosyl donors of opposite configuration. These reactions not only provide a new view of the catalytic capabilities of these supposedly strict hydrolases; they also furnish a basis for defining a detailed mechanism for catalysis. Present results, together with those of several recent studies from this laboratory (especially similar findings obtained with beta-amylase acting on alpha- and beta-maltosyl fluoride (Hehre, E. J., Brewer, C. F., and Genghof, D. S. (1979) J. Biol. Chem. 254, 5942-5950), provide strong new evidence for the functional flexibility of the catalytic groups of carbohydrases.     

A novel one-pot de-blocking and conjugation reaction step leads to process intensification in the manufacture of PEGylated insulin IN-105

Bio-catalytic in vitro multistep reactions can be combined in a single step in one pot by optimizing multistep reactions under identical reaction condition. Using this analogy, the process of making PEGylated insulin, IN-105, was simplified. Instead of taking the purified active insulin bulk powder as the starting material for the conjugation step, an insulin process intermediate, partially purified insulin ester, was taken as starting material. Process intensification (PI) was established by performing a novel de-blocking (de-esterification) of the partially purified insulin ester and conjugation at B-29 Lys residue of B chain with a short-chain methoxy polyethylene glycol (mPEG) in a single-pot reactor. The chromatographic profile at the end of the reaction was found similar irrespective of whether both the reactions were performed sequentially or simultaneously. The conjugated product of interest, IN-105 (conjugation at LysB(29)), was purified from the heterogeneous mixture of conjugated products. The new manufacturing process was deduced to be more simplified and economical in making the insulin conjugates as several downstream purification steps could be circumvented. The physicochemical characteristics of IN-105 manufactured through this economic process was found to be indifferent from the product formed through the traditional process where the conjugation starting material was purified from bulk insulin.     

Group-specific and type-specific gel diffusion precipitin tests for bluetongue virus serotype 20 and related viruses

Using antigens prepared from cell cultures infected by bluetongue (BLU) virus type 20 (BLU-20), and sera from cattle which had recovered from experimental infection by that virus, two distinct precipitin reactions were demonstrated by immunodiffusion. Two distinct gel diffusion precipitin tests were developed based on these reactions. The antigen of one was common to BLU-20 and two other Australian BLU isolates, CSIRO 154 (BLU-21) and CSIRO 156 (BLU-1). It was therefore concluded to be a group-specific test. The antigen of the second appeared to be unique to BLU-20. The test based on this antigen correlated well with the virus neutralization test for BLU-20 and it was therefore concluded to be type-specific. Similar methods applied to a virus of the Palyam (PAL) group demonstrated two precipitin reactions of similar broad (group) and narrow (type) specificity.     

Chemoreceptor function and metabolism of the carotid body in the rat

Using fluorimetric and polarographic determination of cell respiration, metabolic reactions of the carotid body of rats to adequate stimulation have been revealed. Functional relationships of the dose--effect type, which were found for these reactions, may be approximated by equations which are similar to those describing the unconditioned reflexes from the carotid chemoreceptors. However, reactions of the metabolics systems of the carotid body account for the perception of not all chemical stimuli, but only of acids and some alkaloids. These data reveal the heteregeneity of the carotid chemosensory system not only in the amphibians (which was known earlier), but in mammals as well.     

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.     

Demonstration of the isolation of non-infective Alnus crispa var. mollis Fern, nodule endophyte by morphological immunolabelling and whole cell composition studies.

Two filamentous, branched, and septate actinomycetes were isolated from field-collected and from axenic in vitro produced root nodules of Alnus crispa var. mollis Fern. host plant. After their transfer to a chemically defined medium, these nodule isolates could not be distinguished from each other on the basis of morphology, cultural reactions, and whole cell composition and were considered to be the same species. They were morphologically similar to the root nodule endophyte, but were incapable of nodulating aseptic host plants growing in a nitrogen-deficient substrate. Whole cells of the nodule isolates were used for the production of rabbit antibodies. The resulting specific antiisolate antibodies were conjugated with fluorescein isothiocyanate and used in staining tests of the nodule endophyte. The immunofluorescence reactions demonstrated the homology of the nodule isolates with the nodule endophyte. After pectinase degradation of the endophyte capsule, the indirect immunoferritin method corroborated the fluorescent anti-body (FA) staining reactions. There was no antigenic relationship between the nodule isolates and 13 known strains of actinomycetes as determined by the FA techique. Fluorescent antibody reactions of adsorbed conjugates suggested that endophytes of both Alnus crispa var. mollis Fern. and Alnus rugosa (DuRoi) Spreng. root nodules belong to a common serotype. The LL and mesoisomers of diaminopimelic acid were present in similar proportions in the nodule endophyte and in the nodule isolates. Glucose, mannose, and an unknown sugar were the predominant whole cell sugars in the nodule isolates, although trace amounts of arabinose and rhamnose were also displayed. The unknown sugar found in the nodule isolates was also present in trace amounts in the endophyte-suspension hydrolysate.