Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris

An enzyme catalyzing the formation of δ-aminolevulinic acid by transamination of γ,δ-dioxovaleric acid with l-α-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in δ-aminolevulinic acid metabolism in plants is not clearly understood.     

l-Phenylalanine Ammonia-Lyase (Maize): Evidence for a Common Catalytic Site for l-Phenylalanine and l-Tyrosine

l-Phenylalanine ammonia-lyase (E.C. 4.3.1.5) from maize is active with l-tyrosine and l-phenylalanine and exhibits atypical Michaelis-Menten kinetics with both substrates. With phenylalanine as a substrate, the pH optimum is 8.7 and with tyrosine, 7.7. The estimated Km at high substrate concentrations is 0.27 mm for phenylalanine and 0.029 mm for tyrosine. However, the V_max with phenylalanine is eight times higher than the V_max with tyrosine when both are measured at pH 8.7, and 7 times higher when both are measured at their pH optima. The following evidence leads us to the conclusion that there is a common catalytic site for both substrates: (a) It is impossible to appreciably alter the ratio of the two activities during purification and isoelectric focusing. (b) The ratio of the products formed in mixed substrate experiments is in good agreement with the ratio predicted from the estimated Km values. (c) NaBH₄ reduces both activities to the same degree and l-phenylalanine, l-tyrosine, cinnamate, and p-coumarate protect both activities against NaBH₄ reduction to the same degree. In contrast, the enzyme isolated from potato, which does not act on l-tyrosine, is not protected against reduction by either l-tyrosine or p-coumarate. However, both enzymes appear to have a dehydroalanine-containing prosthetic group.     

Inhibition of glycosidases by aldonolactones of corresponding configuration. The specificity of α-l-arabinosidase

1. The previous study (Conchie, Gelman & Levvy, 1967b) of the specificity of β-glucosidase, β-galactosidase and β-d-fucosidase in barley, limpet, almond emulsin and rat epididymis was extended to α-l-arabinosidase. 2. The inhibitory action of l-arabinono-(1→5)-lactone was tested against all four types of enzyme, and α-l-arabinosidase was examined for inhibition by glucono-, galactono- and d-fucono-lactone. 3. In emulsin, the enzyme that hydrolyses β-glucosides, β-galactosides and β-d-fucosides also hydrolyses α-l-arabinosides. Rat epididymis resembles emulsin except that, as already noted, it lacks β-glucosidase activity. 4. In the limpet, α-l-arabinosidase activity is associated with the enzyme that hydrolyses β-glucosides and β-d-fucosides, and not with the separate β-galactosidase. 5. The effects of the different lactones on the barley preparation suggest that α-l-arabinosidase activity is associated with the β-galactosidase rather than with the enzyme that hydrolyses β-glucosides and β-d-fucosides. Fractionation and heat-inactivation experiments indicate that there is also a separate α-l-arabinosidase in the preparation.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the -amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This -amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation.     

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

Inhibition of glycosidases by aldonolactones of corresponding configuration: The C-4- and C-6-specificity of β-glucosidase and β-galactosidase

1. In barley, β-glucosidase and β-galactosidase are separate enzymes. The former also displays β-d-fucosidase activity. 2. In the limpet, Patella vulgata, β-glucosidase activity is associated with the β-d-fucosidase, previously shown to be a separate entity from the β-galactosidase also present. 3. Almond emulsin presents all three activities as a single enzyme. Each is equally inhibited by glucono-, galactono- and d-fucono-lactone. 4. In rat epididymis, there is no significant β-glucosidase activity, nor is there appreciable inhibition of the β-galactosidase and β-d-fucosidase activities of the preparation by gluconolactone.     

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α₄β₄γ₄), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

The C-S Lyases of Higher Plants : Isolation and Properties of Homogeneous Cystine Lyase from Broccoli (Brassica oleracea var botrytis) Buds

Cystine lyase degrades l-cystine by a β-elimination to form cysteine persulfide, pyruvate, and ammonia. This enzyme is common in Brassica sp. and has been purified to homogeneity from extracts of broccoli (Brassica oleracea var botrytis) buds. Two isozymes were separated on DEAE-Fractogel columns and the first peak, cystine lyase I further purified to homogeneity. The purified enzyme had a narrow range of substrate specificity with l-cystine and S-alkyl-l-cysteine sulfoxides being the primary substrates. The K_m for l-cystine was 1.9 millimolar and for S-ethyl-l-cysteine sulfoxide was 15.6 millimolar, suggesting that l-cystine would be preferred in vivo. Using gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the holoenzyme was estimated as 152,000 composed of subunits of approximately 49,000. This strongly suggests the native enzyme is a trimer. The presence of carbohydrate in the native enzyme was detected at the level of 5.8% on a weight basis. Except for the ability to utilize l-cystine as a substrate there are many similarities between cystine lyase I and the alliin lyase of onion (Allium cepa).     

Promotion of seed germination by cyanide

Potassium cyanide at 3 μm to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form β-l cyanoalanine, is active in the seeds. β-l-Cyanoalanine is the most effective of the 23 α-amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from β-cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-¹⁴C is much lower than of cyanide.     

β-Alanine Biosynthesis in Methanocaldococcus jannaschii

One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A K_m of ∼0.80 mM for l-aspartate with a specific activity of 0.09 μmol min⁻¹ mg⁻¹ at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.     

Efficient Synthesis of Eriodictyol from l-Tyrosine in Escherichia coli

The health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration in Escherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3′-hydroxylase (tF3′H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) in E. coli. This allowed the engineered E. coli to produce eriodictyol from l-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol from l-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids from l-tyrosine or even glucose in E. coli.     

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the β-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid –bound FoRham1 were determined, and the active site was identified on the anterior side of the β-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.     

Host-Pathogen Interactions : XXIX. Oligogalacturonides Released from Sodium Polypectate by Endopolygalacturonic Acid Lyase Are Elicitors of Phytoalexins in Soybean

Recent studies have demonstrated that an apparently homogeneous preparation of an α-1,4-d-endopolygalacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max [L.] Merr. cv Wayne) and that this pectin-degrading enzyme released heat-stable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate (KR Davis et al. 1984 Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P-6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of α-1,4-d-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-β-l-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) galactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-α-1,4-d-galactosyluronic acid that contained 4,5-unsaturated galactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants.     

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.     

Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat

1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action.