Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of ³⁵SO₄²⁻ to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33-fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 μm, while for adenosine phosphosulfate, the Km was 1.35 μm. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3′-phosphoadenosine-5′-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with β-glucosidase, β-GIcNAcase, and α-mannosidase, respectively. Swainsonine specifically inhibited α-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited β-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.     

Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2+

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly¹⁵⁹ to Val, Ser²⁰⁸ to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca²⁺ (10 mm) was comparable with MMP-1 (wild type), but in low Ca²⁺ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca²⁺. Varying the concentration of Zn²⁺ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca²⁺ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca²⁺ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca²⁺ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.     

Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by d-galactosone

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [¹⁴C]Uridine labelling of hepatoma cells indicated a division of [¹⁴C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmol×h⁻¹×(kg AS-30D or liver wet wt.)⁻¹. As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of ¹⁴CO₂ into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5′-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37°C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.     

Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of ³⁵SO₄²⁻ into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5′-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of ³⁵SO₄²⁻ into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5′-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO₂ and nitrate assimilation.     

E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (k_obs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of k_obs on TDG concentration gives k_max = 1.6 min⁻¹ and K_1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (K_d ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Yeast Nem1-Spo7 Protein Phosphatase Activity on Pah1 Phosphatidate Phosphatase Is Specific for the Pho85-Pho80 Protein Kinase Phosphorylation Sites

Pah1 is the phosphatidate phosphatase in the yeast Saccharomyces cerevisiae that produces diacylglycerol for triacylglycerol synthesis and concurrently controls the levels of phosphatidate used for phospholipid synthesis. Phosphorylation and dephosphorylation of Pah1 regulate its subcellular location and phosphatidate phosphatase activity. Compared with its phosphorylation by multiple protein kinases, Pah1 is dephosphorylated by a protein phosphatase complex consisting of Nem1 (catalytic subunit) and Spo7 (regulatory subunit). In this work, we characterized the Nem1-Spo7 phosphatase complex for its enzymological, kinetic, and regulatory properties with phosphorylated Pah1. The dephosphorylation of Pah1 by Nem1-Spo7 phosphatase resulted in the stimulation (6-fold) of phosphatidate phosphatase activity. For Pah1 phosphorylated by the Pho85-Pho80 kinase complex, maximum Nem1-Spo7 phosphatase activity required Mg²⁺ ions (8 mm) and Triton X-100 (0.25 mm) at pH 5.0. The energy of activation for the reaction was 8.4 kcal/mol, and the enzyme was thermally labile at temperatures above 40 °C. The enzyme activity was inhibited by sodium vanadate, sodium fluoride, N-ethylmaleimide, and phenylglyoxal but was not significantly affected by lipids or nucleotides. Nem1-Spo7 phosphatase activity was dependent on the concentrations of Pah1 phosphorylated by Pho85-Pho80, Cdc28-cyclin B, PKA, and PKC with k_cat and K_m values of 0.29 s⁻¹ and 81 nm, 0.11 s⁻¹ and 127 nm, 0.10 s⁻¹ and 46 nm, and 0.02 s⁻¹ and 38 nm, respectively. Its specificity constant (k_cat/K_m) for Pah1 phosphorylated by Pho85-Pho80 was 1.6-, 4-, and 6-fold higher, respectively, than that phosphorylated by PKA, Cdc28-cyclin B, and PKC.     

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [γ-³²P]adenosine 5′-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F₁ is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F₁ but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.     

Partial purification and properties of an enzyme from rat liver that catalyses the sulphation of l-tyrosyl derivatives

1. An enzyme that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′[³⁵S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3′-phosphate 5′-sulphatophosphate–phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or −10°C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

Malate dehydrogenase isoenzymes in division synchronized cultures of euglena

Sucrose density gradient centrifugation of broken cell suspensions of autotrophically grown Euglena gracilis Klebs. has allowed the separation of chloroplasts, mitochondria, and peroxisomes. Chlorophyll was taken as a marker for chloroplasts, fumarase and succinate dehydrogenase for mitochondria, and glycolate oxidoreductase for peroxisomes. Peaks of malate dehydrogenase (l-malate-NAD oxidoreductase, EC 1.1.1.37) activity were found in the mitochondrial and peroxisomal fractions. Acrylamide gel electrophoresis showed specific isoenzymes in the mitochondrial and peroxisomal fractions and a third isoenzyme in the supernatant. The mitochondrial isoenzyme which had a Km (oxaloacetate) of 30μm was inhibited by oxaloacetate concentrations above 0.17 mm, an inhibition of 50% being given by 0.9 mm oxaloacetate. The peroxisomal isoenzyme had a Km (oxaloacetate) of 24 μm, was inhibited by oxaloacetate concentrations above 0.13 mm, 50% inhibition being given by 0.25 mm oxaloacetate. Malate dehydrogenase activity in the supernatant did not show inhibition by increasing oxaloacetate concentration, the Km (oxaloacetate) being 91 μm.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.