scholarly journals Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases

1988 ◽  
Vol 250 (1) ◽  
pp. 145-152 ◽  
Author(s):  
L A Kleczkowski ◽  
D D Randall
Keyword(s):  
Substrate Inhibition ◽  
The Novel ◽  
Purification And Characterization ◽  
Leaf Extracts ◽  
Hydroxypyruvate Reductase ◽  
Dependent Activity ◽  
Immunological Comparison ◽  
Spinach Leaf ◽  
Ph Range

A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate ‘leaked’ from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.

scholarly journals Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase

1986 ◽  
Vol 239 (3) ◽  
pp. 653-659 ◽  
Author(s):  
L A Kleczkowski ◽  
D D Randall ◽  
D G Blevins
Keyword(s):  
High Specificity ◽  
Maximal Activity ◽  
Purification And Characterization ◽  
Leaf Extracts ◽  
Hydroxypyruvate Reductase ◽  
Ph Range ◽  
Glyoxylate Reductase ◽  
Spinach Leaves

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.

Purification and characterization of catalase HPII from Escherichia coli K12

1986 ◽  
Vol 64 (7) ◽  
pp. 638-646 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Unusual Structure ◽  
Escherichia Coli K12 ◽  
Molecular Weights ◽  
Ph Range ◽  
Heme Cofactor ◽  
Native Molecular Weight

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90 000 and 92 000. Only a single 92 000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532 000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4–11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

Purification and characterization of catalase-1 from Bacillus subtilis

1987 ◽  
Vol 65 (11) ◽  
pp. 939-947 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Catalase Activity ◽  
Purification And Characterization ◽  
Ph Range ◽  
Sulfhydryl Compounds ◽  
Native Molecular Weight

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395 000. Only one subunit type with a molecular weight of 65 000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5–11 and was only slowly inactivated at 65 °C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.

scholarly journals Purification and characterization of aspartate aminotransferase from the halophile archaebacterium Haloferax mediterranei

1991 ◽  
Vol 278 (1) ◽  
pp. 149-154 ◽  
Author(s):  
F J G Muriana ◽  
M C Alvarez-Ossorio ◽  
A M Relimpio
Keyword(s):  
Aspartate Aminotransferase ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Scanning Calorimetry ◽  
Haloferax Mediterranei ◽  
First Order Kinetics ◽  
Low Salt ◽  
Titration Data ◽  
Ph Range

Aspartate aminotransferase from the archaebacterium Haloferax mediterranei was purified and found to be homogeneous. An average Mr of 66,000 was estimated. The native halophilic transaminase exhibited no maximum absorption at 410 nm, which indicates that the apo form is obtained by our purification procedure, and the molar absorption coefficient at 275 nm in 3.5 M-KCl (pH 7.8) was found to be 78.34 mM-1.cm-1. Plots of titration data show that 1 mol of halophilic aspartate aminotransferase binds 2 mol of pyridoxal 5′-phosphate. The halophilic transaminase behaved as a dimer with two similar subunits and had a maximum activity in the pH range 7.6-7.9 and at 65 degrees C in 3.5 M-KCl. By differential scanning calorimetry, the denaturation temperature of the halophilic holo- and apo-transaminase was determined to be 78.5 and 68.0 degrees C respectively at 3.3 M-KCl (pH 7.8). At low salt concentration the halophilic transaminase was inactivated, following first-order kinetics. The Km values for 2-oxoglutarate and L-aspartate, in 3 M-KCl (pH 7.8), were 0.75 mM and 12.6 mM respectively.

Purification and Characterization of an Extracellular Cold-Active Protease Produced by the Psychrotrophic Bacterium serratia Sp. WJ39

2014 ◽  
Vol 618 ◽  
pp. 330-334 ◽  
Author(s):  
Xiu Ling Ji ◽  
Muhammad Kamran Taj ◽  
Xiao Bo Lu ◽  
Lian Bing Lin ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Residual Activity ◽  
Leather Industry ◽  
Purification And Characterization ◽  
Suitable Candidate ◽  
Optimal Activity ◽  
Sds Page ◽  
Cold Active ◽  
Active Protease ◽  
Ph Range

Proteases have diverse applications in a wide variety of industries, such as in detergent, leather, food, pharmaceutical and silk. The extracellular cold-active protease was purified from the psychrotrophic bacteriumSerratiasp. WJ39 from a meat factory. The protease was cold-active with a molecular mass of 47.6 kDa estimated on SDS-PAGE. It showed an optimal activity at pH of 8 and was stable at pH 6 to 10, while its optimal temperature was 37°C and it was stable at 0-25°C, even remained 35% residual activity at 0°C. The protease was totally inhibited by PMSF which was telling that the purified enzyme was a serine protease. The properties like moderate thermostability, activity in a broad pH range and resistance to metal ions make this enzyme a suitable candidate for the possible use in food and leather industry.

Purification and characterization of a β-1,3-glucanase from the novel mycoparasite Periconia byssoides

2007 ◽  
Vol 29 (4) ◽  
pp. 617-622 ◽  
Author(s):  
Chao Lin ◽  
Jinkui Yang ◽  
Hui Sun ◽  
Xiaowei Huang ◽  
Ruibin Wang ◽  
...  
Keyword(s):  
The Novel ◽  
Purification And Characterization
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