Halvor Aarnes

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Abstract Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg 2+ or Mn 2+ , and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.

Cellulose fibers from endodontic paper points as an etiological factor in postendodontic periapical granulomas and cysts

Histological sections of eight periapical granulomas and cysts developing after conventional endodontic therapy and displaying faintly hematoxylinophilic, birefringent foreign bodies were investigated by light and polarization microscopy, scanning electron microscopy, energy-dispersive X-ray, and chemical analysis. In addition to a variably dense mononuclear infiltrate, the granulomas and cysts revealed varying amounts of giant cells associated with the birefringent foreign bodies. These structures were identified as cellulose fibers, most probably originating from endodontic paper points, which in our opinion can be held responsible for the initiation and perpetuation of chronic postendodontic periapical lesions.

Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.)

Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K+ and the Km value for K+ was observed to be 4.3 mmol l−1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5′-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.

Biosynthesis of threonine from homoserine in pea seedlings: II. Threonine synthase

Summary Threonine synthase was extracted from green shoots of 14-day-old pea seedlings and purified by ammonium sulfate fractionation and DEAE-Sephadex A-50 chromatography. Pyridoxal phosphate was required for maximal activity with purified enzyme. The enzyme was activated in a cooperative manner by low concentrations of S -adenosylmethionine (SAM). The presence of 0.4 mM S -adenosylmethionine lowered the K m value of O -phosphohomoserine (HSP) from 2.2 mM to 0.67 mM and increased the reaction velocity 4-fold or more.

A lysine-sensitive aspartate kinase and two molecular forms of homoserine dehydrogenase from barley seedlings

Abstract A lysine-sensitive aspartate kinase was purified about 27-fold from etiolated barley seedlings. The apparent K m values for l -aspartate and ATP were 6.6 mM and 4.2 mM, respectively. The K i value for l -lysine was estimated to be 0.4 mM. Two molecular forms of homoserine dehydrogenase were partially purified from barley seedlings. The form with the highest molecular weight was inhibited by l -threonine, the other was not. Both molecular forms were inhibited by l -cysteine and l -aspartate.

Homoserine kinase from barley seedlings

Abstract Homoserine kinase was partially purified from 4-day-old etiolated barley seedlings (Hordeum vulgare L.). The enzyme activity required the presence of divalent ions (Mg2+ > Mn2+ > Zn2+ > Co2+). Monovalent cations (K+ or NH4+) were also required for catalytic activity. Homoserine and MgATP protected the enzyme against heat inactivation. The apparent Km-values for homoserine and ATP were 0.4 and 1.0 mM, respectively. The enzyme was not feed-back inhibited by the amino acids threonine, methionine or isoleucine.

Partial purification and characterization of methionine adenosyltransferase from pea seedlings

Abstract Methionine adenosyltransferase was partially purified from green shoots of 14-day-old pea seedlings. Recovery of the enzyme was improved by inclusion of the substrates MgATP and methionine in the extraction medium. Pea methionine adenosyltransferase has an absolute dependence on both divalent (Mg2+ or Mn2+) and monovalent (K+ or NH 4+ cations for activity. The Km value for methionine was 0.4 mM and for MgATP 0.3 mM. The enzyme activity was inhibited by tripolyphosphate and S-adenosyl-methionine.

Reduction of net photosynthesis in oats after treatment with low concentrations of ozone

Oats (Avena sativa L. cv Titus) were exposed to low concentrations of O3 in an assimilation chamber system. Net photosynthesis (net CO2 uptake), measured before and after O3 fumigation, showed significantly different responses for leaves of different age. The oldest active leaf was the most sensitive to O3. Net photosynthesis was depressed after 2 h with 0.075 ppm (150 microg m(-3)) O3. For leaves exposed to 0.150 ppm (300 microg m(-3)) O3 for 2 h, net photosynthesis was reduced significantly for 4 h, after which recovery occurred, nearly reaching the preexposure level 19 h after the exposure. Dark respiration was initially more than doubled after exposure to 0.130 ppm (260 microg m(-3)) O3. There was no visible injury after any of the experiments. The results indicate that O3 may cause crop losses through effects on photosynthesis even in Scandinavia, where a typical O3 episode lasts 1 to 2 h, and the concentration seldom exceeds 0.150 ppm.

Biosynthesis of the thioether cystathionine in barley seedlings

Abstract The regulatory properties of a cystathionine γ-synthase preparation isolated from the first leaf of 4-day-old light-grown barley seedlings were investigated. The enzyme was partially purified by fractionation with ammonium sulfate, heat treatment and gel filtration. The enzyme required O-phosphohomoserine and cysteine for activity. The Km for O-phosphohomoserine (PHS) was approx. 0.13 mM and that for cysteine was about 0.4 mM. The optimal activity of cystathionine γ-synthase appeared at approx. pH 6.8 and the molecular weight estimated by gel filtration was about 180 000. The enzyme activity was inhibited by a wide range of inorganic salts.

Effects of polychlorinated biphenyls on the neutrophil NADPH oxidase system

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.