Indian Journal of Biochemistry & Biophysics

 

CODEN : IJBBBQ  ISSN : 0301-1208

 
Total visitors: 2,385  since 14-06-07

 

 

VOLUME 44

NUMBER 4

AUGUST 2007

 
CONTENTS

 

 

Papers

 

Site-directed mutagenesis of conserved Thr407, Asp433, and Met464 residues in small subunit of Escherichia coli g-glutamyltranspeptidase

 

197

       Huei-Fen Lo, Long-Liu Lin*, Pei-Jing Chen and Wei-Mou Chou
Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

 

204

       Daling Zhu, Aihua Li, Jianguo Wang*, Ming Li and Taozhen Cai

Azadirachta indica leaf extract modulates initiation phase of murine forestomach tumorigenesis

 

209

        Subhash Chander Gangar and Ashwani Koul*

 

 

 

Metabolism of arachidonic acid in sheep uterus: In vitro studies

216

        A Sai Padma*, S Agarwal, D Bharat Reddy, T Sandeep Prasad and
P Reddanna

 

 

 

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars

 

223

        Kamaljit Kaur, Anil K Gupta and Narinder Kaur*

 

 

 

Effect of UV-B and high visual radiation on photosynthesis in freshwater
(Nostoc spongiaeforme) and marine (Phormidium corium) cyanobacteria

 

231

        Rupali Bhandari and Prabhat Kumar Sharma*

 

 

 

Vibrational dynamics and heat capacity in polyglycine II

240

        Abhishek Kumar Mishra, Mahendra Singh, Poonam Tandon* and Vishwambhar Dayal Gupta

 

 

 

Notes

 

Purification and properties of an extracellular pectin lyase produced by the strain of Penicillium oxalicum in solid-state fermentation

 

247

        Sangeeta Yadav* and N V Shastri

 

 

 

Purification and some properties of carbonic anhydrase from Elephas trogontherii (Steppe elephant) bone

 

252

        Yaşar Demir, Hayrunnisa Nadaroğlu and Nazan Demir*

 

 

 

 

Modulation of DMBA-induced biochemical changes by organoselenium compounds in blood of rats

 

257

        I Ozdemir, Z Selamoglu, B Ates, Y Gok and I Yilmaz*

 

 

 

Instructions to Authors

260

 

 

 

 

——————

*Author for correspondence

 

 

 

PAPERS

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 197-203

 

Site-directed mutagenesis of conserved Thr407, Asp433 and Met464 residues in small subunit of Escherichia coli g-glutamyltranspeptidase

Huei-Fen Lo1, Long-Liu Lin2,*, Pei-Jing Chen3 and Wei-Mou Chou3,*

1Department of Food and Nutrition, Hungkuang University, Shalu, Taichung City, Taiwan

2Department of Applied Chemistry, National Chiayi University, Chiayi City, Taiwan

3Graduate Institute of Biotechnology, National Chiayi University, Chiayi City, Taiwan

Received 10 April 2007; revised 20 July 2007

Sequence comparison showed that residues Thr407, Asp433, and Met464 in the small subunit of Escherichia coli g-glutamyltranspeptidase (EcGGT) were conserved in the aligned enzymes. In this study, we further investigated the functional significance of these conserved residues by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 cells and purified to near homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown no GGT activity under enzyme assay conditions and activity staining. Furthermore, mutations on these residues impaired the capability of autocatalytic processing of the enzyme. Based on these observations, it is concluded that these residues play an important role in the enzyme maturation.

Keywords: Escherichia coli, g-Glutamyltranspeptidase, Site-directed mutagenesis, Autocatalytic processing

*E-mail: llin@mail.ncyu.edu.tw

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 204--208

 

Cloning, expression and characterization of aerolysin fromAeromonas hydrophila in Escherichia coli

Daling Zhua,b, Aihua Lia, Jianguo Wanga, * , Ming Lia and Taozhen Caia

aThe Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

bGraduate School of the Chinese Academy of Sciences, Beijing 100039, China

Received 02 December 2006; revised June 2007

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc.A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W)of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli bl21. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His·Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.

KeywordsAeromonas hydrophila, Cloning, aerA gene, Prokaryotic expression, Recombinant protein AerA-W, Hemolytic activity

*Email: wangjg@ihb.ac.cn

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 209-215

 

Azadirachta indica leaf extract modulates initiation phase of murine forestomach tumorigenesis

Subhash Chander Gangar and Ashwani Koul*

Department of Biophysics, Basic Medical Sciences Block, Panjab University, Chandigarh 160 014, India

Received 02 March 2007; revised 09 July 2007

The effects of aqueous Azadirachta indica leaf extract (AAILE) on benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis, B(a)P-DNA adduct formation and certain parameters of carcinogen biotransformation system in mice have been reported earlier from our laboratory. In this study, the effects of AAILE on the enzymes of B(a)P biotransformation, which play crucial role in initiation of chemical carcinogenesis ¾ aryl hydrocarbon hydroxylase (AHH) and uridinediphosphoglucuronosyltransferase (UDP-glucuronosyltransferase) have been evaluated in murine forestomach and liver. In addition, lipid peroxidation (LPO) levels in forestomach as well as liver and the activities of tissue injury marker enzymes ¾ lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase in the serum have also been evaluated. Oral administration of AAILE (100 mg/kg body wt for 2 weeks) reduces the AHH activity and enhances the UDP-glucuronosyltransferase activity in both the tissues, suggesting its potential in decreasing the activation and increasing the detoxification of carcinogens. The LPO levels decrease upon AAILE treatment in the hepatic tissue, suggesting its anti-oxidative and hence anti-carcinogenic effects. Non-significant alterations have been observed in tissue injury marker enzymes upon AAILE treatment, suggesting its safety at the given dose . In conclusion, AAILE appears to modulate initiation phase of carcinogenesis and may be suggested as safe and an effective agent for chemoprevention.

Keywords: Aryl hydrocarbon hydroxylase; Azadirachta indica; Benzo(a)pyrene; Lipid peroxidation; Tissue injury marker enzymes; UDP-glucuronosyltransferase

*Email :ashwanik@pu.ac.in, drashwanikoul@yahoo.co.in

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 216-222

 

Metabolism of arachidonic acid in sheep uterus: In vitro studies

A Sai Padmaa*, S Agarwalb, D Bharat Reddyb, T Sandeep Prasada and P Reddannab

a Department of Biochemistry, Bharatiya Vidya Bhavan’s Vivekananda College, Sainikpuri, Secunderabad 500 094, India

bSchool of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

Received 24 January 2007; revised 22 May 2007

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins ¾ PGF2α and prostacyclin derivative 6-keto PGF1α. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

Keywords: Arachidonic acid metabosism, Lipoxygenase, Cyclooxygenase, Prostaglandins, HPETEs and HETEs

*E-mail: saipadmabhavans@yahoo.co.in

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 223-230

 

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars

 

Kamaljit Kaur, Anil K Gupta and Narinder Kaur*

Department of Biochemistry and Chemistry, Punjab Agricultural University, Ludhiana 141 004, India

Received 09 February 2007; revised 14 May 2007

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (a and b amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the a -amylase activity in the endosperm of seedlings of both the cultivars, but increased a and b amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.

Keywords:    Triticum aestivum L., Water deficit, Acid invertase, Alkaline invertase, a and b -Amylase, Sucrose phosphate synthase, Sucrose synthase, Sucrose, Starch

*E-mail: nkaur @rediffmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 231-239

 

Effect of UV-B and high visual radiation on photosynthesis in freshwater
(Nostoc spongiaeforme) and marine (Phormidium corium) cyanobacteria

Rupali Bhandari and Prabhat Kumar Sharma*

Department of Botany, Goa University, Goa 403 206, India

Received 11 September 2006; revised 17 May 2007

Human activity is causing depletion of ozone in stratosphere, resulting in increased UV-B radiation and global warming. However, impact of these climatic changes on the aquatic organism (especially marine) is not fully understood. Here, we have studied the effect of excess UV-B and visible radiation on photosynthetic pigments, fatty acids content, lipid peroxidation, nitrogen content, nitrogen reductase activity and membrane proteins, induction of mycosporine-like amino acids (MAAs) and antioxidant enzymes superoxide dismutase (SOD) and ascorbate peroxidase (APX) in freshwater (Nostoc spongiaeform) and marine (Phormidium corium) cyanobacteria. UV-B treatment resulted in an increase in photosynthetic pigments in Nostoc and decrease in Phormidium, but high light treatment caused photobleaching of most of the pigments in both the species. Unsaturation level of fatty acids of both total and glycolipids remained unchanged in both the cyanobacteria, as a result of
UV-B and high light treatments. Saturated fatty acids of total and glycolipids declined slightly in Nostoc by both the treatments, but remained unchanged in Phormidium. No changes in the unsaturated lipid content in our study probably suggested adaptation of the organism to the treatments. However, both treatments resulted in peroxidation of membrane lipids, indicating oxidative damage to lipids without any change in the level of unsaturation of fatty acid in the cell membrane. Qualitative and quantitative changes were observed in membrane protein profile due to the treatments. Cyanobacteria were able to synthesize MAAs in response to the UV-B treatment. Both treatments also increased the activities of SOD and APX. In conclusion, the study demonstrated induction of antioxidants such as SOD and APX under visible light treatment and screening pigment (MAAs) under UV-B treatment, which might protect the cyanobacteria from oxidative damage caused by high light and UV-B radiation.

Keywords: Antioxidant enzymes, Cyanobacteria, Fatty acids, High light, Mycosporine-like amino acids, Photosynthetic active radiation, Photosynthetic pigments, Ultraviolet-B radiation

*Email: prabhat_k_sharma@rediffmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 240-246

 

Vibrational dynamics and heat capacity in polyglycine II

Abhishek Kumar Mishraa, Mahendra Singhb, Poonam Tandona* and Vishwambhar Dayal Guptaa

aDepartment of Physics, University of Lucknow, Lucknow 226 007, India

 bBrahmanand P.G. College , Kanpur 208 004, India

Received29 September 2006;revised 21 June 2007

The earlier works on the vibrational dynamics of polyglycine II (PG II) suffer from several infirmities, which not only suppress the dynamical nature of normal modes, but also lead to several incorrect assignments and interactive constants of the potential field. In this study, we have re-examined the phonon dispersion profiles of PG II using Higgs method for evaluation of phase-related normal modes and have attempted to remove the infirmities, as far as possible. The Wilson’s GF matrix method combined with the Urey-Bradley force field has been used for normal mode analysis. This potential field leads to correct assignments of Raman, infrared and inelastic neutron scattering frequencies. Characteristic features of the dispersion curve (v versusd /p plot), such as repulsion and regions of high density-of-states have been interpreted. In addition, the heat capacity as a function of temperature has been obtained via density-of-states. It agrees well with the experimental data and is being reported for the first time.

Keywords: Polyglycine II, Density-of-states, Phonon dispersion, Heat capacity, Urey-Bradley force field

*E-mail: poonam_tandon@yahoo.co.uk

 

NOTES

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 247-251

 

Purification and properties of an extracellular pectin lyase produced by the strain of Penicillium oxalicum in solid-state fermentation

Sangeeta Yadav*1 and N V Shastri

Department of Biochemistry, RTM Nagpur University, Amravati Road, Nagpur 440 010, India

Received 09 May 2006; revised 07 May 2007

A pectin lyase (PNL, EC 4.2.2.10) produced extracellularly by the strain of Penicillium oxalicum in solid-state fermentation medium containing deoiled mandarin orange peel meal was purified to apparent homogeneity by a protocol that included ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatography. The enzyme had molecular mass of 50 kD, as determined by SDS- PAGE and showed optimum pH and temperature at 8.0 and 50° C respectively. It had an isoelectric point (pI) of 5.0 and showed a Km of 1.1 mg/ml of citrus pectin. The enzyme was strongly inhibited by Mo4+, Ag+ and Pb2+ and also by polyphenolic compounds, in particular tannic acid.

Keywords: Pectins, Pectin lyases, Pectin transeliminases, Penicillium oxalicum, Solid-state fermentation

*Email: sangeeta_rahul@rediffmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp. 252-256

 

Purification and some properties of carbonic anhydrase from Elephas trogontherii
(Steppe elephant) bone

Yaşar Demir1, Hayrunnisa Nadaroğlu2 and Nazan Demir 3*

1Department of Chemistry, Faculty of Education
2Department of Food Technology, Oltu Profession High Scholl
3Department of Chemistry, Faculty of Science, Atatürk University, 25240, Erzurum, Turkey

 

Received 07 November 2006; revised 18 July 2007

Four isoenzymes of carbonic anhydrase (CA) were purified from Elephas trogontherii (steppe elephant) bone (approx 0.3-0.5 million years old) from different locations (outer peripheral, cytosolic, inner peripheral and integral) using Sepharose 4B-L-tyrosine sulphanilamide affinity chromatography and their kinetics properties were investigated and compared with known CA isoenzymes. The purification degree of CAs was monitored by SDS-PAGE. Purification fold for outer peripheral, inner peripheral, cytosolic and integral CA was 395.6, 652.8, 1091 and 429.3 and the molecular mass (as determined by gel filtration chromatography) was 37, 36, 35, and 39 kDa, respectively.The optimal temperature for isozymes was 10-20, 30, 30 and 60° C and optimal pH was between 7.5-11, 7.5-10, 7.5-10 and 7.5 respectively. Km values (at optimum pH and 20° C) for p-nitrophenyl acetate as substrate were 4.83, 6.80, 4.525 and 3.86 mM and the Vmax values for the same substrate were 0.00097, 0.0149, 0.00249 and 0.00072 μmol/L*min, respectively. I50 values of isoenzymes for the inhibitors of CA ¾ sulphanilamide, KSCN, acetazolamide and NaN3 were also determined.

Keywords: Elephas trogontherii, Bone, Carbonic anhydrase, Kinetics properties, Steppe elephant

*E-mail: demirn@yahoo.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, August 2007, pp.257-259

 

Modulation of DMBA-induced biochemical changes by organoselenium compounds in blood of rats

I Ozdemir1, Z Selamoglu2, B Ates1, Y Gok1 and I Yilmaz1*

1Department of Chemistry and 2Department of Biology, Inonu University, 44280 Malatya, Turkey

 

Received 25 April 2006; revised 19 July 2007

The protective role of two synthetic organoselenium compounds 1-isopropyl-3-methylbenzimidazole-2-selenone (SeI) and 1, 3-di-p-methoxybenzylpyrimidine-2-selenone (SeII) was examined against the 7,12-dimethylbenz[a]anthracene (DMBA)-induced changes in biochemical parameters in blood of rats. Albino Winstar rats (150-200 g body wt) were treated with single dose of DMBA (50 mg/kg body wt) and organoselenium compounds (25 m mol/kg) for 4 weeks at two days internal. Blood was taken from the anaesthetized rats ventricle from their hearts for biochemical analysis. Administration of DMBA resulted in elevation of urea, uric acid and creatinine levels as well as AST, ALT and LDH activities and decrease in levels of total proteins, albumin and globulin. SeI and SeII caused a significant (p<0.05) decrease in urea, uric acid and creatinine levels and alanine aminotransferase (ALT); aspartate aminotransferase; (AST) and lactate dehydrogenase (LDH) activities and significantly increased the levels of total protein and albumin (p<0.05). These organoselenium compounds are likely to be beneficial in human health.

Keywords:             Biochemical parameters, 7,12-Dimethylbenz [a]anthracene or DMBA, Oxidative stress, Organoselenium compounds

*E mail: iyilmaz@inonu.edu.tr

 

 

AUTHOR INDEX

 

 


Agarwal S                           216

Ates B                                 257

 

Bhandari R                         231

 

Cai T                                   204

Chen P J                             197

Chou W M                        197

 

Demir N                             252

Demir Y                             252

 

Gangar S C                       209

Gok Y                                257

Gupta A K                       223

Gupta V D                        240

 

Kaur K                             223

Kaur N                             223

Koul A                             209

 

Li A                                  204

Li M                                 204

Lin L L                             197

Lo H F                             197

 

Mishra A K                    240

 

Nadaroğlu H                  252

 

Ozdemir I                        257

 

Prasad T S                     216

 

Reddanna P                   216

Reddy D B                      216

 

Sai Padma A                  216

Selamoglu Z                  257

Sharma P K                    231

Shastri N V                    247

Singh M                         240

 

Tandon P                       240

 

Wang J                          204

 

Yadav S                         247

Yilmaz I                          257

 

Zhu D                            204