Nkat Mg-1 Research Articles

Enzymatic Profile of 'Willamette' Raspberry Leaf and Fruit Affected by Prohexadione-Ca and Young Canes Removal Treatments.

The influence of growth regulator prohexadione-Ca (ProCa) concurrently with young canes removal on the modification of photosynthetic pigments content and antioxidant enzymes (peroxidase, POD; catalase, CAT; polyphenol oxidase, PPO; superoxide dismutase, SOD) activities in leaves and fruits of raspberry (Rubus idaeus L.) cultivar 'Willamette' was studied. ProCa increased while canes removal decreased chlorophylls and carotenoids content compared to control. POD, CAT, and PPO activities in leaves after removal of young canes were higher compared to control (2-4 times) which was visually confirmed for POD by isoelectrofocusing. Removal of young canes slithly increased, while ProCa significantly enhanced SOD activity in leaves compared to control (475.10 and 218.38 nkat mg-1 prot, respectively). Pattern of SOD activity in fruit was similar as in leaf with substantial increase compared to control (about 15 times). Combination of implemented measures increased activity of all enzymes in the leaves and fruits. Our study could provide a better knowledge of the ProCa and canes removal influences on the action of enzymes in order to regulate their activities in fruit products.

Sucrose transport across the vacuolar membrane of Ananas comosus.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In Ananas comosus L. (Merr.) (pineapple), a widely cultivated bromeliad that exhibits crassulacean acid metabolism (CAM), much of the carbohydrate synthesized during the daytime appears to accumulate as soluble sugars in the vacuole. To investigate the mechanism of sugar transport into the vacuole, microsomal extracts were prepared from deacidifying leaves harvested during Phase III of the CAM cycle. The vesicle preparations exhibited features expected for a fraction highly enriched in vacuolar membranes (tonoplast), i.e. the ATPase activity of 16 ±2nkat mg-1 protein was inhibited 96% by 50 mm KNO3, an inhibitor of vacuolar ATPases, and was only 7% inhibited by 100μm NaN3 plus 100μm Na3VO4, inhibitors of mitochondrial and plasma membrane ATPases, respectively. Further, the microsomal ATPase activity showed a pH optimum between 7.0 and 8.0, typical of a vacuolar ATPase. When presented with Mg-ATP, vesicles established H+ gradients that could be maintained for at least 25 min. The vesicles were able to take up [14C]sucrose from an external medium. Sucrose uptake exhibited saturable kinetics with an apparent Km of 50 m sucrose and apparent Vmax of 171 ± 5 pkat mg-1 protein. Sucrose uptake was not dependent upon, nor stimulated by, Mg-ATP, suggesting that the mechanism of sucrose transport into the vacuole in A. comosus does not involve H+-coupled cotransport. However, the initial rates of sucrose uptake from the external medium were stimulated when vesicles were preloaded with sucrose. This trans-stimulation is consistent with characteristics expected for a sucrose uniporter capable of operating in an exchange mode. It is proposed that the accumulation of glucose and fructose in leaf vacuoles of Ananas during the light period involves at least two steps - transport of sucrose into the vacuole by a mechanism exhibiting characteristics of a sucrose uniporter, followed by cleavage of sucrose by a vacuolar acid invertase to form glucose and fructose.

Some fungi express β-1,3-glucanases similar to thaumatin-like proteins

Mycelial clarified homogenates from Lentinus edodes, Agaricus bisporus, Rhizoctonia solani and extracts from commercial sources of lysing enzymes from Rhizoctonia solani or Irpex lacteus contained enzymes of ca 25 kDa hydrolyzing polymeric carboxy-methylated (CM)-pachyman with an in-gel β-1,3-glucanase assay. Proteins were purified from Rhizoctonia solani, Lentinus edodes and Irpex lacteus and their Nterminal amino acid sequences showed homology to thaumatin-like (TL) proteins. As with plant TL proteins with β-1,3-glucanase activity, little or no activity was observed on oligomeric laminarin by the in-gel hydrolase assay. The fungal TL proteins varied in specific activity from 60 to 760 nkat mg-1 with CM-pachyman as substrate and were thus more active than most plant TL proteins. Thin-layer chromatography indicated that the fungal TL proteins generated various oligoglucosides from an endo β-1,3-glucanase activity. The TL proteins were detected in several, but not all, anastomosis groups of Rhizoctonia solani. The purified Rhizoctonia solani TL protein was the most potent enzyme, inhibiting the growth of Saccharomyces cerevisiae when compared to two fungal or three plant TL proteins. Moreover, the Rhizoctonia TL protein was able to hydrolyze live Saccharomyces yeast cells. This is the first report of fungal TL proteins with endo-β-1,3-glucanase activity and strong binding to polymeric β-1,3-glucans.

Some thaumatin-like proteins hydrolyse polymeric beta-1,3-glucans.

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase.

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13.6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.

Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum.

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein.

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl2. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 microM chloramphenicol, 10 microM 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits alpha (49 kDa), beta (39 kDa) and gamma (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of alpha and beta but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric alpha beta gamma-structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the beta-subunits.

Extracellular proteinase from Brevibacterium linens. Purification and characterization

Brevibacterium linens produces proteolytic enzymes in cultivation broth. One of them was purified by application of a two step procedure involving ultrafiltration and molecular sieving. The isolated enzyme hydrolyzed natural substrates: casein, hemoglobin, ovalbumin and bovine serum albumin with rations 22.2, 3.5, 1.6, 1.2 nkat mg-1, respectively. For casein the apparent Km 1.8 mg ml-1 was determined. The proteinase is fairly stable at pH 8.0 at 30 °C. Short term incubation at 50 °C denaturated the proteinase. SDS PAGE revealed an Mr of the proteolytic enzyme about 52 000 to 55 000.

Partial Purification and Characterization of Fructokinase Activity from Barley Leaves

Summary Barley (Hordeum vulgare L.) leaf extracts contained a nonspecific hexokinase that was almost exlusivelylocalized in the particulate fraction. In addition, three soluble isoforms of fructose-specific kinase activity were separable by ion exchange chromatography and these were designated fructokinase-Ia, -Ib and -II, based upon elution order. Final 6700-, 1500- and 800-fold purification resulted in specific activities of 233.8, 51.8 and 60.1 nkat mg-1 protein for the three fructokinase isoforms, respectively. None of the isoforms were homogeneous. Native enzyme molecular weights were 37, 37 and 73kg mol-1 for fructokinase-I a, -I b and -II, respectively. All three isoforms had broad pH optima with maxima near pH 8.0 and were activated by K+ (Ka = 0.6 to 1.3 molm-3). Apparent Km values for fructose and MgATP were between 100 and 200 mmolm-3. Substrate inhibition in the range of 5 to 25 mol m-3 fructose was severe for fructokinase-I a and -Ib but only slight with fructokinase-II. All three fructokinase isoforms utilized UTP, but with apparent Km values about 5- to 10-fold higher than for ATP. Therefore, it is likely that ATP is the natural substrate for barley leaf fructokinases in vivo.

Genetical variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum

Glutamate dehydrogenase (GDH) is the key enzyme of ammonium assimilation by ectomycorrhizal fungi. Its activity might be use as a criterion to select mycelia capable of enhancing the nitrogen nutrition of the host plants. Genetical variability of the GDH activity of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi was studied in an attempt to determine if this enzyme activity could be improved by way of chromosomal genetics. The activity of 11 wild strains was compared with that of 70 mycelia obtained as the progeny of a laboratory fruiting strain HC1. These 70 mycelia were 20 monokaryons (5 for each mating type) and the 50 synthesized dikaryons obtained from all the compatible fusions between these monokaryons. The specific GDH activity of the 11 wild strains ranged from 1.5 to 11.6 nkat mg-1 fungal protein. The activity of the monokaryotic progeny of the HC1 strain was, on average, three times lower (2.85 n kat mg-1 fungal protein) than that of the parental dikaryon. In contrast, synthesized dikaryons originating from these monokaryons were very variable and had an average values similar to that of the parental dikaryon (9.1 nkat mg-1 fungal protein). Eighteen of these synthesized dikaryons contained an activity higher than that of the original HC1 strain. The variation of the GDH activity of these dikaryons involves additive and non additive (interactive) components, each of them accounting for ca. 50% of the genetical variation. The non additive variation could not be explained by a model involving only dominance. These results are discussed with reference to the genetical improvement of mycorrhizal fungi in order to enhance nitrogen nutrition of the host plants.