Indian Journal of Biochemistry & Biophysics

CODEN: IJBBBQ  ISSN: 0301-1208

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

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VOLUME 46

NUMBER 3

JUNE 2009

CONTENTS

Papers
Endopeptidases of Bacillus subtilis IBTC-3 and B. alcalophilus PB92 in synthesis of precursors of biologically active peptides	213
Agnieszka E Głowacka, Mirosława H Szczęsna-Antczak*, Małgorzata Piotrowicz-Wasiak & Tadeusz Z Antczak
Biochemical characterization and some biological properties of the phosphodiesterase I purified from Agistrodon bilineatus venom	221
Saad S M Al-Saleh*, Sami Ullah Khan & Mohammad Ashraf
Characterization of arylsulphatase A in a 70 kDa protein isolated from goat spermatozoa having Na+, K+-ATPase inhibitory activity	230
Tushar K Dhara, Madhumouli Chatterjee, Rabindranath Bera & Parimal C Sen*
Cellular AATF gene encodes a novel miRNA that can contribute to HIV-1 latency	237
Deepak Kaul* & Aashiq Hussain
Antioxidant effect of ethanolic extract of Piper betle Linn. (Paan) on erythrocytes from patients with HbE-beta thalassemia	241
Phalguni Srimani, Goutam Mandal, Sudipto Ganguly, Piu Saha, Rupashree Sen, Rajib De, Arnab Chatterjee, Maitrayee Bhattacharyya, Utpal Chaudhuri, Susanta K Bandyopadhyay & Mitali Chatterjee*
Evaluation of antioxidant potential of Clitoria ternata L. and Eclipta prostrata L.	247
D Bhaskar Rao*, Ch Ravi Kiran, Y Madhavi, P Koteswara Rao & T Raghava Rao
Photoinhibition and photosynthetic acclimation of rice (Oryza sativa L. cv Jyothi) plants grown under different light intensities and photoinhibited under field conditions	253
Janet Vaz & Prabhat Kumar Sharma*
Buffering capacity and membrane H+ conductance of protease producing facultative alkaliphilic bacterium Bacillus flexus from mangrove soil	261
P Kannan, S Ignacimuthu * & M Gabriel Paulraj
Notes
Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA	266
T N Jayashri, R Anuradha & N S Punekar *
Antioxidant status in polycystic end-staged renal diseased patients and anti-hemolytic effect of Boerhaavia diffusa	269
K Sathya Priya, V Vijayachandrika & C S Parameswari *
Instructions to Authors	273

——————

*Author for correspondence

AUTHOR INDEX

Al-Saleh S S M	221
Antczak T Z	213
Anuradha R	266
Ashraf M	221
Bandyopadhyay S K	241
Bera R	230
Bhattacharyya M	241
Chatterjee A	241
Chatterjee M	230
Chatterjee Mitali	241
Chaudhuri U	241
Dhara T K	230
Głowacka A E	213
Goutam Mandal	241
Hussain A	237
Ignacimuthu S	261
Jayashri T N	266
Kannan P	261
Kaul D	237
Khan S U	221
Kiran Ch R	247
Madhavi Y	247
Parameswari C S	269
Paulraj M G	261
Piotrowicz-Wasiak M	213
Piu Saha	241
Punekar N S	266
Rajib De	241
Rao D B	247
Rao P K	247
Rao T R	247
Rupashree Sen	241
Sathyapriya K	269
Sen P C	230
Sharma P K	253
Srimani P	241
Sudipto Ganguly	241
Szczęsna-Antczak M H	213
Vaz J	253
Vijayachandrika V	269

PAPERS

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.213-220

Endopeptidases of Bacillus subtilis IBTC-3 and B. alcalophilus PB92 in synthesis of precursors of biologically active peptides

Agnieszka E Głowacka, Mirosława H Szczęsna-Antczak*, Małgorzata Piotrowicz-Wasiak and Tadeusz Z Antczak

Institute of Technical Biochemistry, Department of Biotechnology and Food Sciences,
Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland

Received 10 July 2008; revised 14 May 2009

Two endopeptidases (from Bacillus subtilis IBTC-3 and from B. alcalophilus PB92-commercial preparation) efficiently synthesized amino acid esters (NAc-Tyr-OEt and NAc-Phe-OEt) and dipeptides (NAc-Tyr-Gly-NH₂ and NAc-Tyr-Arg-NH₂) in organic solvent/water systems. The rate of NAc-Tyr-OEt synthesis mediated by the native subtilisin IBTC-3 was maximum (0.23 Umg^-1) in ethanol/5-7% w/v water system, while the highest activity of the freeze-dried enzyme (0.18 Umg^-1) was achieved, when water content was 9-10% w/v. The preferred system for dipeptide synthesis (using NAc-Tyr-OEt as acyl donor) by both the enzymes was acetonitrile/4% w/v water. In this system, the maximum yield of NAc-Tyr-GlyNH₂ was 71 and 80% and that of NAc-Tyr-Arg-NH₂ was 53 and 40% for subtilisin IBTC-3 and peptidase PB92, respectively. In contrast to the peptidase PB92, the subtilisin efficiently catalyzed esterification of NAc-Tyr with 1-butanol and isopropanol.

Keywords: Subtilisin, Alkaline peptidase PB92, Immobilization, Amino acid esters, Peptides, Non-aqueous enzymology

*E-mail: mirszcz@p.lodz.pl

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.221-229

Biochemical characterization and some biological properties of the phosphodiesterase I purified from Agistrodon bilineatus venom

Saad S M Al-Saleh^1*, Sami Ullah Khan¹ and Mohammad Ashraf ²

¹Dept. of Clinical Lab. Sciences, College of Applied Medical Sciences, King Saud University P.O. Box 10219,
Riyadh 11433, Saudi Arabia

²Dept. of Pharmacy, Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

Received 30 May 2008; revised 05 March 2009

The venom phosphodiesterase I (PDE-I, EC 3.1.4.1) is useful in the elucidation of the structure and nucleotide sequence of nucleic acids. In the present study, PDE-I was purified from Agistrodon bilineatus venom by preparative native-PAGE. A single protein band was observed in analytical native-PAGE. The enzyme also gave a single band in SDS-PAGE with a molecular mass of 140 kDa. The position of the band was not altered in the presence of β-mercaptoethanol, suggesting the protein did not contain subunits. The enzyme was free from 5’-nucleotidase and alkaline phosphatase activities. It showed a broad optimum pH range (9.0-11.0), whereas the optimum temperature was found to be 60⁰C, with activity decreasing at >65⁰C. Energy of activation (E_a) was calculated to be 0.31. The PDE-I was a glycoprotein having 14% of carbohydrate content. The V_max, K_m, K_cat and K_sp values of the enzyme were 3.85 μM/min/mg, 8.3 × 10^{-3 M, 23s-1} and 46.4 M^-1 Min^-1 respectively. Cysteine caused a non-competitive inhibition with a K_i 6.3 × 10⁻³ M (IC₅₀ of 1.6 mM), whereas ADP caused a competitive inhibition having K_i 0.8 × 10⁻³ M (IC₅₀ 5.4 mM). Glutathione, o-phenanthroline, zinc and EDTA inhibited the enzyme activity, whereas Mg²⁺ slightly potentiated the activity. The enzyme hydrolyzed thymidine 5’-monophosphate p-nitro-phenyl ester most readily (10-fold), while 3’-5’-cAMP was least readily hydrolyzed substrate. The enzyme up to 4.0 mg/Kg i.p was not lethal in mice. It exhibited an anticoagulant effect, and increased the normal clotting time of normal citrated human plasma, whereas the crude venom showed strong coagulant effect. The above results showed that the A. bilineatus PDE-I was very similar to that isolated from other snake venoms. The purification procedure described here is simple, rapid and reproducible and may prove useful to isolate pure protein for investigation into the contribution of this enzyme to the biological activities of A. bilineatus venom and PDE-I insight, in general.

Keywords: Agistrodon bilineatus, Snake, Venom, Phosphodiesterase I

*Email: khansamiullah@hotmail.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.230-236

Characterization of arylsulphatase A in a 70 kDa protein isolated from goat spermatozoa having Na⁺, K⁺-ATPase inhibitory activity

Tushar K Dhara, Madhumouli Chatterjee, Rabindranath Bera and Parimal C Sen*

Division of Molecular Medicine, Bose Institute, P -1/12, C.I.T Scheme –VII-M, Kolkata 700 054, India

Received 07 July 2008; revised 10 March 2009

A protein having inhibitory effect on Na⁺, K⁺-ATPase as well as showing arylsulphatase A activity (ASA) was isolated from the cytosolic fraction of goat spermatozoa and characterized biochemically. The molecular mass of the protein was found to be 70 kDa (P70) on 10% SDS-PAGE after 35% ammonium sulphate precipitation, followed by hydroxyapatite column chromatographic separation. The isoelectric point (pI) of the protein was found to be 4.9. The sequencing results of first ten N-terminal amino acid residues of protein showed 100%, 90%, and 80% homology with N-terminal 18-27 amino acid residues of mice, pig and human testicular ASA, respectively. The optimum pH, temperature and incubation time for maximum ASA activity of the protein was 5.5, 37°C and 30 min respectively. The ASA activity of protein and AS from a commercial source was studied with respect to the sensitivity to different metal ions, vanadate, carbonyl compounds and ascorbate. Inhibition of AS activity of P70 by silver nitrate suggested that it was related to ASA. Comparable effects of different polyunsaturated fatty acids (eicosapentaenoic and docosahexaenoic acids) and purified anti P70-antibody on P70 and AS from commercial source were observed. The findings suggested that protein was novel in nature, having both regulatory and catalytic functions and showed similarities with the ASA reported from different sources.

Keywords: Arylsulphatase A, Goat spermatozoa, Inhibitor of Na⁺, K⁺-ATPase, 70 kDa Protein

*Email: parimalsen.boseinst@gmail.com; parimal@bic.boseins.ernet.in

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.237-240

Cellular AATF gene encodes a novel miRNA that can contribute to HIV-1 latency

Deepak Kaul* and Aashiq Hussain

Molecular Biology Unit, Deptt. of Experimental Medicine & Biotechnology,
Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India

Received 12 July 2008, revised 15 April 2009

HIV-1 encoded microRNA hiv1-miR-H1 is known to induce CD4⁺ lymphopenia through its ability to downregulate cellular AATF gene. The present study directed to examine the target sites of this miRNA on AATF gene revealed the existence of a novel miRNA designated as hmiR-che-1 which had the inherent capacity to target HIV-1 genome especially regions coding for hiv1-miR-H1 as well as Vpr gene. Further, the expression of AATF gene coupled with its encoded microRNA hmiR-che-1 exhibited characteristic antagonism with the expression of hiv1-miR-H1 within the lymphocytes, derived from asymptomatic as well as symptomatic AIDS subjects. Based upon these observations, we propose that the widely recognised HIV-1 latency in CD4⁺ T-lymphocytes may arise, because of the orchestrated balance that may exist between the expression levels of hiv1-miR-H1 and hmiR-che-1 within lymphocytes infected with HIV-1

Keywords: miRNA, hmiR-che-1, AATF gene, Lymphocytes, AIDS

*E-mail: dkaul_24@hotmail.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.241-246

Antioxidant effect of ethanolic extract of Piper betle Linn. (Paan) on erythrocytes from patients with HbE-beta thalassemia

Phalguni Srimani, Goutam Mandal, Sudipto Ganguly, Piu Saha, Rupashree Sen, Rajib De^#, Arnab Chatterjee^#,
Maitrayee Bhattacharyya^#, Utpal Chaudhuri^#, Susanta K Bandyopadhyay and Mitali Chatterjee*

Dept. of Pharmacology, Institute of Postgraduate Medical Education and Research, 244 B, Acharya JC Bose Road, Kolkata 700 020

^#Institute of Haematology and Transfusion Medicine, 88, College Street, Kolkata, 700 073, India

Received15 December 2008; revised 21 May 2009

HbE-beta thalassemia is caused by an interaction between HbE and defective b globin gene of thalassemia. Repeated blood transfusions cause an iron overload, triggering an enhanced generation of free radicals. In the present study, the anti-oxidant property of ethanolic extract of the leaves of Piper betle Linn. (PB) was evaluated in the erythrocytes from patients with HbE-beta thalassemia. In patients with HbE-beta thalassemia (n = 30) and age- and sex-matched healthy individuals
(n = 30), the baseline level of reactive oxygen species (ROS) and free radical scavenging activity in the erythrocytes was measured by flow cytometry using dihydrodichlorofluorescein diacetate (H₂DCFDA), in terms of the geometric mean fluorescence channel (GMFC). The baseline generation of ROS was significantly higher in the erythrocytes from patients with HbE-beta thalassemia, as compared to healthy volunteers, the GMFC being 67.20 ± 4.64 vs. 23.03 ± 1.88 (p<0.001), which was effectively decreased by PB. Similarly, H₂O₂ (0.5-1.0 mM) induced a higher increase in the GMFC in the erythrocytes from patients with HbE-beta thalassemia, as compared to controls which was effectively reduced by PB. Taken together, PB showed promising anti-oxidant activity against the erythrocytes from patients with HbE-beta thalassemia.

Keywords: HbE beta thalassemia, Reactive oxygen species (ROS), Piper betle Linn., Anti-oxidant, Erythrocytes

*E-mail: ilatim@vsnl.net

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.247-252

Evaluation of antioxidant potential of Clitoria ternata L. and Eclipta prostrata L.

D Bhaskar Rao*, Ch Ravi Kiran, Y Madhavi, P Koteswara Rao and T Raghava Rao

Department of Biochemistry, Andhra University, Visakhapatnam 530 003, India

Received 22 October 2008; revised 25 March 2009

Free radical-mediated oxidative stress is believed to be the primary cause of many disorders, such as cardiovascular diseases, brain dysfunction, cataract, diabetes mellitus, arthritis, cancer, ageing etc. In treatment of these diseases, antioxidant therapy has gained an utmost importance in the recent years. Current research is now directed towards finding naturally occurring antioxidants of plant origin. In Indian system of medicine, Clitoria ternata L. and Eclipta prostrata L. are the important medicinal plants, which have a wide range of applications. In the present study, the antioxidant potential of aqueous extracts of C. ternata and E. prostrata was evaluated by determining the levels of enzymatic and non-enzymatic antioxidants. In vitro antioxidant capacity was also determined using different assays and the results were compared with standard antioxidants such as butylated hydroxy toluene (BHT), ascorbic acid and rutin. Our results showed that both plant extracts possessed significant levels of enzymatic and non-enzymatic antioxidants and also exhibited antioxidant capacity. However, C. ternata showed higher levels of enzymatic and non-enzymatic antioxidants, as compared to E. prostrata. In addition, the antioxidant capacity of C. ternata was observed to be significant as compared to E. prostrata.

Keywords: Free radicals, Antioxidants, Clitoria ternata, Eclipta prostrata, Radical scavenging activity

*E-mail: trrao_au@yahoo.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.253-260

Photoinhibition and photosynthetic acclimation of rice (Oryza sativa L. cv Jyothi) plants grown under different light intensities and photoinhibited
under field conditions

Janet Vaz and Prabhat Kumar Sharma*

Dept. of Botany, Goa University, Goa 403206, India

Received 30 June 2008; revised 16 April 2009

Thirty-days old rice (Oryza sativa L. cv. Jyothi) plants grown under the greenhouse (150-200 µmol m^-2 s^-1) or shade (600-800 µmol m^-2s^-1) were exposed to 7 days of full sunlight and compared with plants grown under direct sunlight (1200-2200 µmol m^-2s^-1).Transfer of greenhouse and shade plants to full sunlight for a day resulted in a decline in their photosynthetic efficiency (F_v/F_m) and an increase in non-photochemical quenching (qN). The decline in F_v/F_m was much greater in transferred greenhouse plants (33%) as compared to transferred shade-plants (20%). Sun-plants did not show much variation in the F_v/F_m ratio (4%) from their predawn measurements (control). The sun-grown plants showed a higher pool of xanthophyll pigments (violaxanthin + antheraxanthin + zeaxanthin). Transfer of greenhouse and shade-plants to full sunlight resulted in an increase in lutein, Chl a/b ratio, antheraxanthin (A) and zeaxanthin (Z) content. Increase in A and Z was correlated with the increase in the qN. The increase in the A and Z content was due to increase in the activity of violaxanthin de-epoxidase. Greenhouse and shade plants on exposure to sunlight showed an increase in lipid peroxidation (LPO). Prolonged exposure of greenhouse and shade plants up to 7 days resulted in recovery of the F_v/F_m, an increase in Z and A and a decline in the LPO. The study demonstrated that rice plants grown at lower light intensities initially underwent photoinhibitory damage on exposure to full sunlight, but were able to acclimate to the high irradiance by dissipating the excess light through various mechanisms such as an increase in lutein, high Chl a/b ratio and xanthophyll cycle, suggesting use of energy dissipation as a mechanism of protection against high irradiance, but to different extent and to some extent by different processes. The study was unique, as plants were grown and photoinhibited under natural conditions rather than the artificial light, as was the case in most of the studies so far. Results showed better adaptation of high-light grown plants and suggested role for chl a/b ratio and lutein, in addition to xanthophylls cycle in shade plants. Low-light grown plants could also completely adapt to full level of sunlight within 3 days of the treatment and xanthophylls cycle (measured as V, A and Z) and activity of de-epoxidase seemed to be important in this adaptation.

Keywords: Energy dissipation, Lipid peroxidation, Non-photochemical quenching, Photosynthetic acclimation, Photosynthetic efficiency, Rice plants, Sunlight, Xanthophyll cycle, Chlorophyll a/b ratio, Violaxanthin de-epoxidase

*E-mail: prabhat_k_sharma@rediffmail.com

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.261-265

Buffering capacity and membrane H⁺ conductance of protease producing facultative alkaliphilic bacterium Bacillus flexus from mangrove soil

P Kannan^#, S Ignacimuthu* and M Gabriel Paulraj

Entomology Research Institute, Loyola College, Chennai 600 034, India

Received 23 July 2008; revised 15 April 2009

A facultative alkaliphilic protease-producing gram-positive rod-shaped bacteria (EMGA 5) was isolated from mangrove soil and confirmed as Bacillus flexus by the 16S rDNA sequence. Buffering capacity and membrane H⁺ conductance of this alkaliphilic isolate were investigated for the cells grown at pH 7.2 and 10.5 using acid pulse technique. Suspensions of B. flexus cells grown in poly peptone yeast glucose medium at pH 10.5 exhibited higher cytoplasmic membrane buffering capacity values (70 µmol H⁺/pH unit/mg protein at pH 9.9) than the cells grown at pH 7.2 (41 µmol H⁺/pH unit/mg protein at pH 9.9). B. flexus grown aerobically at pH 7.2 showed higher H⁺ conductance values than the cells grown at pH 10.5 (0.032 µmol H⁺/s/pH unit/mg protein at pH 9.9 and 0.028 µmol H⁺/s/pH unit/mg protein at pH 9.8, respectively). The present study revealed that the buffering capacity and membrane H⁺ conductance of the B. flexus isolates were influenced by pH of the medium.

Keywords: Bacillus flexus, Buffering capacity, Facultative alkaliphile, Membrane H⁺ conductance, Protease

*Email: entolc@hotmail.com

NOTE

Indian Journal of Biochemistry & Biophysics

Vol. 46, June 2009, pp.266-268

Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA

T N Jayashri, R Anuradha and N S Punekar*

Biotechnology Group, School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India

Received 02 December 2008; revised 08 April 2009

A useful variant of PCR technique was devised to generate full-length Aspergillus niger arginase cDNA for expression. Briefly, a 450 bp amplicon was first constructed through overlap extension PCR (OE-PCR) by splicing in a 101 nucleotide long single-stranded megaprimer, facilitated by inclusion of an additional, shorter forward primer in the reaction. The amplicon was suitably cloned into pBlueScript to obtain pArg440 and the insert sequenced. The full-length arginase cDNA was subsequently assembled in pArg440 and moved into pET23a for heterologous expression. An interesting feature of this strategy was not to stoichiometrically incorporate the oligonucleotide megaprimer, but use it only as an early template. This OE-PCR strategy to utilize long single-stranded megaprimer may prove handy in terms of efficiency, yield and sequence choice.

Keywords: Single-stranded megaprimer, Overlap extension PCR, Splicing, Aspergillus niger, Arginase

*Email: nsp@iitb.ac.in

Indian Journal of Biochemistry & Biophysics

Vol. 46, April 2009, pp.269-272

Antioxidant status in polycystic end-staged renal diseased patients and antihemolytic effect of Boerhaavia diffusa

K Sathyapriya, V Vijayachandrika and C S Parameswari*

Post-Graduate Department of Biochemistry, Bharathi Women’s College (Autonomous), Prakasam Salai, North Chennai 600 108

Received 18 December 2008; revised 05 May 2009

Chronic renal failure (CRF) induces anaemia by shortening the life-span of erythrocytes, due to an increase in oxidative stress, which is considered to be one of the major risk factors in CRF patients undergoing hemodialysis. In the present study, the antioxidant status of the end-staged renal disease (ESRD) patients was investigated. The antihemolytic activity of Boerhaavia diffusa on the erythrocytes of the patients was also studied. Protein, lipid peroxides (LPO), reduced glutathione (GSH) levels and glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities were measured in the hemolysate from 55 polycystic ESRD patients (Group II) and compared with normal subjects (Group I). The antioxidant status was found to be significantly reduced in the patients as compared to normal healthy volunteers, due to increased oxidative stress. Also, aqueous extract of B. diffusa showed a significant antihemolytic activity on the erythrocytes of the polycystic ESRD patients.

Keywords:Hemodialysis, Erythrocytes, Boerhaavia diffusa, End-staged renal disease, Antioxidant status, Antihemolytic activity

*Email: cspbiochem@gmail.com