Indian Journal of Biochemistry & Biophysics

                                      CODEN: IJBBBQ  ISSN: 0301-1208

                                  http://www.niscair.res.in; http://nopr.niscair.res.in

 

VOLUME46

NUMBER5

OCTOBER2009

 
CONTENTS

 

 

Papers

 

Short nucleotide sequences signal spliceosomal binding in nucleic acids

353

        K Rupa Reddy and Chanchal K Mitra*

 

 

 

Purification and characterization of mycoferritin from Aspergillus flavus MTCC 873

360

        V Vakdevi, R B Sashidhar and Vijay Deshpande*

 

 

 

Efficient and rapid purification of lentil a-galactosidase by affinity precipitation with

alginate

366

        Evran Bıçak Çelem, Sharon Sibel Bolle and Seçil Önal*

 

 

 

Protective role of Cassia auriculata leaf extract on hyperglycemia-induced oxidative

stress and its safety evaluation

371

        Shipra Gupta, Suman Bala Sharma*, Krishna Madhava Prabhu and Surendra Kumar Bansal

 

 

 

Effect of gallic acid on alkaline phosphatase and peptidase activities in rat intestine

378

        Nidhi Mahajan and Akhtar Mahmood*

 

 

 

Oxidation of oxymyoglobin by poplar plastocyanins a and b

383

        Petya K Christova, Anthony A Donchev, Alexandra Ch Shosheva, Vladimir I Getov and Mitko I Dimitrov*

 

 

 

Kinetics of oxidation of adenosine by tert-butoxyl radicals: Protection and repair by

chlorogenic acid

389

        G Vijayalakshmi, M Adinarayana* and P Jayaprakash Rao

 

 

 

Analysis of comparative efficiencies of different transformation methods of E. coli using

two common plasmid vectors

395

        Aryadeep Roychoudhury*, Supratim Basu and Dibyendu N Sengupta

 

 

 

Notes

 

Partial purification and some properties of α-amylase from Bacillus subtilis KIBGE-HAS

401

        Saeeda Bano, Shah Ali Ul Qader*, Afsheen Aman and Abid Azhar

 

 

 

Effect of mercury ion on the stability of the lipid-protein complex of isolated chloroplasts

405

        Sunakar Panda* and Sumita Panda

 

 

 

Instructions to Authors

409

 

 

——————

*Author for correspondence


 

                      AUTHOR INDEX

 

 


Adinarayana M

389

Aman A

401

Azhar A

401

 

 

Bano S

401

Bansal S K

371

Basu S

395

Bolle S S

366

 

 

Çelem E B

366

Christova P K

383

 

 

Deshpande V

360

Dimitrov M I

383

Donchev A A

383

 

 

Getov V I

383

Gupta S

371

 

 

Mahajan N

378

Mahmood A

378

Mitra C K

353

 

 

Önal S

366

 

 

Panda Sumita

405

Panda Sunakar

405

Prabhu K M

371

 

 

Rao P J

389

Reddy K R

353

Roychoudhury A

395

 

 

Sashidhar R B

360

Sengupta D N

395

Sharma S B

371

Shosheva A Ch

383

 

 

Ul-Qader S A

401

 

 

Vakdevi V

360

Vijayalakshmi G

389



PAPERS

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.353-359

 

Short nucleotide sequences signal spliceosomal binding in nucleic acids

K Rupa Reddy and Chanchal K Mitra*

Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India

Received 25 April 2009; revised 01 September 2009

We have explored the region around the splice sites of the human intron and exons from the exon-intron database (EID) and located a number of short 6-nucleotide and 7-nucleotide sequences that are relatively common in the regions. These short sequences, we expect play an important role in the selection of the appropriate splicing process. We propose that the external signals via short recognition sequences play the deterministic role in the actual splicing process. We have obtained 50 such sequences each from the exon and intron from the beginning and from the ending and noted a number of common features.

Keywords: Splicing sites, Alternative splicing, Short sequences, Spliceosome binding

*E-mail: c_mitra@yahoo.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.360-365

 

Purification and characterization of mycoferritin from
Aspergillusflavus MTCC 873

V Vakdevi, R B Sashidhar and Vijay Deshpande*

Department of Biochemistry, University College of Science, Osmania University, Hyderabad, 500 007, Andhra Pradesh, India

Received 01 June 2009; revised 25 September 2009

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.

Keywords: Induction, Mycoferritin, N-terminal amino acid sequence, Cross-reactivity, Aflatoxin, Aspergillus flavus MTCC 873, Aspergillus parasiticus 255

*E-mail: vdpande123@rediffmail.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.366-370

 

Efficient and rapid purification of lentil a-galactosidase by affinity precipitation with alginate

Evran Bıçak Çelem, Sharon Sibel Bolle and Seçil Önal*

Department of Biochemistry, Faculty of Science, Ege University, Bornova-İzmir, Turkey

Received 05 June 2009; revised 23 September 2009

a-Galactosidase (a-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40oC and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65oC and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl-a-D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of a-galactosidase.

Keywords: Affinity macroligand, Affinity precipitation, Alginate, a-Galactosidase, Lentil seed, Purification

*Email: secil.onal@ege.edu.tr

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.371-377

 

Protective role of Cassia auriculata leaf extract on hyperglycemia-induced oxidative stress and its safety evaluation

Shipra Gupta1, Suman Bala Sharma1*, Krishna Madhava Prabhu1 and Surendra Kumar Bansal2

1Department of Biochemistry, University College of Medical Sciences (University of Delhi), Delhi 110095, India

2Department of Biochemistry, Vallabhbhai Patel Chest Institute (University of Delhi), Delhi 110007, India

Received 23 May 2009; revised 16 September 2009

Cassia auriculata L. (Caesalpiniaceae) is widely used from the ancient period to treat diabetes mellitus. In the present study, the antioxidant activity of C. auriculata aqueous leaf extract (CLEt) was evaluated in streptozotocin-induced mild diabetic (MD) and severe diabetic (SD) rats. A short-term toxicity assessment was also conducted in healthy rats to examine toxic effects of the extract. Oral administration of CLEt to MD and SD rats (100, 200 and 400 mg/kg body weight per day for a period of 21 days) produced significant fall in fasting blood glucose (FBG) in a dose-dependent manner. Treatment with the extract (400 mg/kg) showed significant reduction in serum levels of thiobarbituric acid reactive substances (TBARS) and oxidized low-density lipoprotein (OxLDL) in both MD and SD rats. The antioxidant defense system was also found to be improved in CLEt-treated (400 mg/kg) MD and SD rats, as revealed by significant increase in activities of erythrocyte’s antioxidant enzymes i.e. superoxide dismutase (SOD) and catalase (CAT) with a concomitant elevation in erythrocyte’s reduced glutathione (GSH) content. Moreover, there were no toxic signs in rats treated with high doses of the extract (1000 and 2000 mg/kg body weight per day for 21 days). Blood glucose, hepatic and renal function parameters in these rats were found within normal limits. Phytochemical screening of CLEt revealed the presence of alkaloids, flavonoids, saponins, tannins and cardiac glycosides with antihyperglycemic and antioxidant properties. This study suggests that CLEt possesses potent antioxidant activity along with antihyperglycemic potential, hence protective against diabetic complications.

Keywords: Cassia auriculata, Diabetes mellitus, Streptozotocin, Phytochemicals, Antioxidant activity, Toxicity assessment

*E-mail: drsbs08@yahoo.in

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.378-382

 

Effect of gallic acid on alkaline phosphatase and peptidase activities in rat intestine

Nidhi Mahajan and Akhtar Mahmood*

Department of Biochemistry, Panjab University, Chandigarh, 160014, India

Received 08 May 2009; revised 17 August 2009

Gallic acid is a normal constituent of many edible foods, thus directly interacts with epithelial tissue in intestine. In the present study, the effect of gallic acid on intestinal alkaline phosphatase (IAP) and peptidase activities in rat intestine was evaluated. Gallic acid (0.27-0.5 mM) inhibited activities of leucine aminopeptidase (LAP) and g-glutamyl transpeptidase (g-GTP) by over 90%, compared to controls in rat intestine. In contrast, 0.1-0.6 mM gallic acid either had no effect or stimulated the activity of IAP in rat intestine. The observed inhibition of peptidases by gallic acid was reversible in nature. Kinetic analysis revealed no change in Vmax of LAP (0.42-0.44 units/mg protein) and g-GTP (0.22-0.24 units/mg protein), while the values of apparent Km were increased 6-7 fold, exhibiting competitive-type of enzyme inhibition by gallic acid. The values of Ki for LAP and g-GTP were 0.037 mM and 0.017 mM, respectively. These observations indicate that gallic acid is a potent inhibitor of brush border peptidases, and thus may interfere in the digestion and absorption of proteins in the intestine.

Keywords: Leucine aminopeptidase, g-Glutamyl transpeptidase, Intestinal alkaline phosphatase, Enzyme inhibition, Gallic acid, Rat intestine, Brush border membrane

*E-mail: akhtarmah@yahoo.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.383-388

 

Oxidation of oxymyoglobin by poplar plastocyanins a and b

Petya K Christova1, Anthony A Donchev2, Alexandra Ch Shosheva2, Vladimir I Getov2
and Mitko I Dimitrov2*

1Institute of Organic Chemistry, 2Institute of Biophysics, Bulgarian Academy of Sciences, Sofia-1113, Bulgaria

Received 29 September 2008; revised 03 August 2009

Oxidation of oxymyoglobin [MbO2 (Fe2+)] by isoplastocyanins a (PCa) and b (PCb) was experimentally investigated and the corresponding redox reaction was modeled using the physicochemical parameters of the isoforms to study the effect of the dimorphism. The kinetic curve of oxidation of MbO2 (Fe2+) by oxidized PCa [PCa(Cu2+)] and PCb [PCb(Cu2+)] and the pH-dependence of the rate constant k1 were determined. In the range of pH 4.8-9.0, PCb reacts with higher k1, compared with PCa. For example, at pH 7.0, k1(PCb) = 4 × 102 M-1s-1, whereas k1(PCa) = 2 × 102 M-1s-1. The observed values of ΔE0 for the reaction pairs Mb-PCa and Mb-PCb were -304 mV and -319 mV, respectively. The effect of the ionic strength (µ) on the rate of the electron transfer was also studied. It was found that: (i) the net charge Z1 of PCa and PCb fully corresponds to that calculated by their primary structures and Z2 of Mb corresponds to that calculated by its titration curve; (ii) the ln k as function of √¯µ was similar for both PCa and PCb; (iii) the curve of the reaction PCb  Mb (pH 7.0) was shifted towards higher values of k, in agreement with the larger net negative charge of PCb; and (iv) the character of the electrostatic interactions remained unchanged by a replacement of PCa by PCb and by the change of pH from 7.0 to 4.8.

Keywords: Dimorphism, Photosynthesis, Electron transfer, pH-dependence, Ionic strength dependence

*E-mail: mitkod@bio21.bas.bg

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.389-394

 

Kinetics of oxidation of adenosine by tert-butoxyl radicals: Protection and repair by chlorogenic acid

G Vijayalakshmi, M Adinarayana* and P Jayaprakash Rao

Department of Chemistry, Osmania University, Hyderabad, 500 007, India
*PG College of Science, Saifabad, Osmania University, Hyderabad, 500 004, India

Received 16 July 2008; revised 10 August 2009

The rates of oxidation of adenosine and chlorogenic acid by tert-butoxyl radicals (t-BuO×) were studied by measuring the absorbance of adenosine at 260 nm and chlorogenic acid at 328 nm spectrophotometrically. t-BuO× radicals were generated by the photolysis of tert-butyl hydroperoxide (t-BuOOH) in presence of tert-butyl alcohol to scavenge OH. radicals. The rates and the quantum yields (f) of oxidation of chlorogenic acid by t-BuO×radicals were determined in the absence and presence of varying concentrations of adenosine. An increase in the concentration of adenosine was found to decrease the rate of oxidation of chlorogenic acid, suggesting that adenosine and chlorogenic acid competed for t-BuO. radicals. From competition kinetics, the rate constant of chlorogenic acid reaction with t-BuO× was calculated to be 3.20 ´ 109 dm3 mol-1 s-1. The quantum yields (fexpt) were calculated from the experimentally determined rates of oxidation of chlorogenic acid under different experimental conditions. Assuming that chlorogenic acid acts as a scavenger of t-BuO× radicals only, the quantum yields (fcal) were theoretically calculated. fexpt and fcal values suggested that chlorogenic acid not only protected adenosine from t-BuO× radicals, but also repaired adenosine radicals, formed by the reaction of adenosine with t-BuO× radicals.

Keywords:             Chlorogenic acid, Adenosine repair, t-BuO. radicals, Oxidation

*E-mail: adinarayana_mundra@live.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.395-400

 

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors

Aryadeep Roychoudhury*1, Supratim Basu2 and Dibyendu N Sengupta2

1Department of Botany, Plant Molecular Biology and Biotechnology Laboratory, University of Calcutta,
35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India
2Department of Botany, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Kolkata 700009, West Bengal, India

Received 13 April 2009; revised 04 September 2009

The efficiencies of different transformation methods of E. coli DH5α strain, induced by several cations like Mg2+, Mn2+, Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

Keywords: Calcium chloride, E. coli, Ethanol, Heat shock, Microwave, Plasmid DNA, Transformation efficiency

*E-mail: aryadeep.rc@gmail.com

 

 

NOTES

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.401-404

 

Partial purification and some properties of α-amylase from Bacillus subtilis KIBGE-HAS

Saeeda Bano1, Shah Ali Ul Qader2*, Afsheen Aman2 and Abid Azhar2

1Pharmaceutical Research Centre, PCSIR Laboratories Complex, Karachi, Pakistan

2The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan

Received 10 October 2008; revised 07 September 2009

An extracellular α-amylase from Bacillus subtilis KIBGE-HAS was partially purified by ultrafiltration and ammonium sulphate precipitation with 19.2-fold purification and specific activity of 4195 U/mg. The enzyme showed relatively high thermostability and retained 62% of its activity when kept at 70°C for 15 min. α -Amylase was highly stable at -18°C and loss of activity was very low during stability study. Metal ions like Mn2+, Ca2+, Co2+, K+, Mg2+, and Fe3+ activated the enzyme, while Hg2+ Ba2+, Cu2+, Na+ and Al3+ strongly inhibited the activity. The α-amylase was highly stable in various surfactants and detergents. In the presence of surfactants such as SDS and Triton X-100 the enzyme activity was found 2.9 and 1.8-fold higher respectively than control. The non-ionic detergents (Tween 20 and Tween 80) exhibited slightly inhibitory effect on the enzyme activity.

Keywords: α-Amylase, Bacillus sp., Surfactant, Metal ions, Thermal stability

*E-mail: madar_chem@yahoo.com

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 46, October 2009, pp.405-408

 

       Effect of mercury ion on the stability of the lipid-protein complex of isolated chloroplasts

 

Sunakar Panda* and Sumita Panda

Laboratory of Environmental Biochemistry, P.G. Department of Chemistry, Berhampur University,
Bhanjabihar, Berhampur 760007, Dist. Ganjam, Orissa, India

 

Received 02 February 2009; revised 26 August 2009

Mercury is known to interact with different parts of living systems causing serious biochemical and physiological disorder. In order to know the effect of mercury (Hg2+) ion on chloroplasts, the cell free organelle are incubated in an isotonic buffer medium in presence of mercury ion. The metal ion is found to induce membrane lipid peroxidation, loss of photosynthetic pigments and degradation of proteins. Such degradation brings about a drastic modification of lipid-protein organization of chloroplasts as reflected from a blue shift of absorption peaks and lowering of chlorophyll-a fluorescence intensity. The detrimental effect of Hg2+ ion has been explained in terms of direct binding with lipid-protein complex of photosynthetic membrane. Such a binding of metal ion exposes the lipid-protein complex for an easier entry and attack of reactive oxygen species (ROS) generated during incubation of chloroplasts in light and dark, thereby resulting in higher disorganization, which is evident from cation- induced changes in absorption and emission characteristics of the organelle.

Keywords: Mercury toxicity, Chloroplast, Reactive oxygen species, Photosynthetic assembly

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