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Indian Journal of Biochemistry & Biophysics

 

ISSN : 0301-1208

CODEN : IJBBBQ  

VOLUME 41

NUMBER 4

AUGUST 2004

CONTENTS

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

141

Sun-Young An, Min Ok, Ji-Youn Kim, Moon-Sun Jang, Young-Su Cho, Yong-Lark Choi, Cheorl-Ho Kim and Young-Choon Lee*

 

 

Biochemical characterization of a protein of albumin multigene family from serum of African catfish Clarias gariepinus Bloch

148

Absar-ul Hasnain*, S Hasan Arif, Riaz Ahmad, Mumtaz Jabeen and
M Mushahid Khan

 

 

Partial purification of chlorophyll degrading enzymes from Cavendish banana (Musa Cavendishi)

154

Machhindra T Janave* and Arun Sharma

 

Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+- and Ca2+-ATPases by modulator proteins isolated from rat brain cytosol

162

Dipankar Bhattacharyya, Subho Ghosh, Madhumouli Chatterjee, Arindam Basu and Parimal C Sen*

 

 

Antitumour activity of Annona squamosa seed extracts is through the generation of free radicals and induction of apoptosis

167

B V V Pardhasaradhi, Madhurima Reddy, A Mubarak Ali, A Leela Kumari and Ashok Khar*

 

 

Spectroscopic studies on the interaction of riboflavin with bovine serum albumin

173

B P Kamat, J Seetharamappa* and M B Melwanki

 

Notes

 

Rationalization of physico-chemical properties of 5, 6-diarylthiazolo [3,2-b]-1, 2, 4-triazoles towards cyclooxygenase-2 (COX-2) inhibition: A QSAR approach

179

E Manivannan, S Prasanna and S C Chaturvedi*

 

A study of aromatic hydrogen bonds of peptides with aromatic amino acid side-chains

184

A Nallini, K Saraboji and M N Ponnuswamy*

Effect of aspartame on circadian oscillations of calcium and inorganic phosphorus in rats

188

P Rajasekar, T Manivasagam and P subramanian*

 

Protein profiles of two rice varieties by 2-D gel electrophoresis under moisture stress

191

Arti Chandra and Aruna Tyagi*

 

Instructions to Authors
Announcements 138,197
Author Index

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 141-147

 

Cloning, high-level expression and enzymatic properties of an intracellular
serine protease from Bacillus sp. WRD-2

Sun-Young An, Min Ok, Ji-Youn Kim, Moon-Sun Jang, Young-Su Cho,

Yong-Lark Choi, Cheorl-Ho Kim1 and Young-Choon Lee*

 

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45°C. It was stable at pH from 7.5 to 11.0 and below 50°C.

 

Keywords: Bacillus, cloning, overexpression, intracellular serine protease, purification, recombinant enzyme.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 148-153

 

Biochemical characterization of a protein of albumin multigene family from serum of
African catfish Clarias gariepinus Bloch

Absar-ul Hasnain*, S Hasan Arif, Riaz Ahmad, Mumtaz Jabeen and M Mushahid Khan

 

A monomorphic albumin-like protein (CfSA) has been purified to homogeneity from the serum of African air-breathing catfish Clarias gariepinus Bloch. It has a molecular mass of @70 kD and shows a lesser electrophoretic mobility than human serum albumin (HSA) in native gels. The protein exhibits cross-reactivity against rabbit anti-HSA serum and shows considerable similarity with HSA in secondary structure, however, with some differences, as indicated by a slight shift in the peaks around 267 nm and 278 nm and the absence of shoulders at 276 and 283. A certain degree of similarity also exists between their tertiary structures with respect to aromatic asymmetric environment as indicated by far-UV CD spectra and the visible range CD spectra of bilirubin complexes. CfSA-bilirubin complex is mainly characterized by bisignate CD Cotton effects (CDCEs), having minima and maxima wavelengths at 406 and 486 nm, respectively and unlike HSA, it shows prominent additional maxima around 426 nm. Based on the number of sulfhydryls, CfSA is in the rank of advanced teleosts. The occurrence of albumin in C. gariepinus in relation to the evolutionary dichotomy of albumin and other members of its multigene family in class Pisces has been discussed.

 

Keywords:    Biochemical characterization, ligand transport, catfish serum albumin (CfSA), human serum albumin (HSA), lipoprotein, α-foetoprotein, sulfhydryls, circular dichroism, bilirubin binding, helix topography, Clarias gariepinus.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 41, August 2004, pp. 154-161

 

Partial purification of chlorophyll degrading enzymes from Cavendish banana (Musa Cavendishi)

Machhindra T Janave* and Arun Sharma

 

Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30°C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways ─ chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from ‘green-ripe’ bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from ‘green-unripe’ and ‘yellow-ripe’ bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a single major band, resulting in almost a homogenous preparation in a single step. Fraction IV catalyzed dechelation of Mg by Mg-dechelatase, while fraction VI catalyzed the formation of 132-OH-Chl a by chlorophyll oxidase. Chlorophyll oxidase activity was stimulated by linolenic acid, indicating involvement of lipoxygenase in oxidative Chl degradation, thereby resulting in the formation of 132-OH-Chl a as product. The results show the presence of various enzymes of chlorophyllase and chlorophyll oxidase pathways in soluble enzyme fraction.

 

Keywords:   Cavendish banana, Musa cavendishi, stay-green fruits, green-ripe, green-unripe, yellow-ripe, chlorophyll degrading enzymes, Mg-dechelatase, chlorophyllase, pheophorbide a oxygenase, red fluorescent catabolite (RFC) reductase, chlorophyll oxidase.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 41, August 2004, pp. 162-166

 

Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+- and Ca2+-ATPases by modulator proteins isolated from rat brain cytosol

Dipankar Bhattacharyya, Subho Ghosh, Madhumouli Chatterjee,

Arindam Basu and Parimal C Sen*

 

Two proteins of molecular mass 13 kDa, a specific inhibitor of Na+, K+-ATPase and another of 12 kDa, which can distinguish between Ca2+, Mg2+- and Ca2+-ATPase activities have been obtained from the pooled fractions isolated from rat brain, using Sephadex G-100 chromatography. In order to determine the key step(s), which is affected by the modulators, we have designed an in vitro experiment of phosphorylation and dephosphorylation of these ATPases in the absence and presence of the modulators. The results suggest that the phosphorylation step of Mg2+-independent Ca2+-ATPase is inhibited, while in Mg2+-dependent Ca2+-ATPase, the dephosphorylation step is stimulated by the modulators. The findings support our earlier observation that the modulators are able to distinguish between Mg2+-independent and dependent Ca2+-ATPases activities.

 

Key words: Phosphorylation, dephosphorylation, Ca2+, Mg2+- and Ca2+-ATPases, modulator proteins, rat brain cytosol

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 167-172

 

Antitumour activity of Annona squamosa seed extracts is through the generation
of free radicals and induction of apoptosis

B V V Pardhasaradhi, Madhurima Reddy, A Mubarak Ali,

A Leela Kumari and Ashok Khar*

 

The plant, Annona squamosa traditionally known as custard apple possesses potent bioactive principles in all its parts. The effect of aqueous and organic extracts from defatted seeds of A. squamosa was studied on a rat histiocytic tumour cell line, AK-5. Both the extracts caused significant apoptotic tumour cell death with enhanced caspase-3 activity, down regulation of antiapoptotic genes Bcl-2 and BclXL, and enhanced the generation of intracellular ROS, which correlated well with the decreased levels of intracellular GSH. In addition, DNA fragmentation and annexin-V staining confirmed that the extracts induced apoptosis in tumour cells through the oxidative stress. Aqueous extracts of A. squamosa seeds possessed significant antitumor activity in vivo against AK-5 tumor.

 

Key words: Annona squamosa, reactive oxygen species (ROS), GSH, antitumour activity, apoptosis, Bcl-2, seed extract

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 173-178

 

Spectroscopic studies on the interaction of riboflavin with bovine serum albumin

B P Kamat, J Seetharamappa* and M B Melwanki

 

The mechanism of interaction of riboflavin (RF) with bovine serum albumin (BSA) using fluorometric and circular dichroism (CD) methods has been reported. The association constant (K) for RF-BSA binding shows that the interaction is non-covalent in nature. Stern-Volmer analysis of fluorescence quenching data shows that the fraction of fluorophore (BSA) accessible to the quencher (RF) is close to unity, indicating that both tryptophan residues of BSA are involved in the interaction. The high magnitude of rate constant for quenching kq (1013 M-1s-1) indicates that RF binding site is in close proximity to tryptophan residue of BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of RF to BSA predominantly involves the formation of hydrophobic bonds. Binding studies in the presence of a hydrophobic probe 8-anilino-1-naphthalene sulphonic acid, sodium salt (ANS) showed that RF and ANS do not share common sites in BSA. The small decrease in critical micellar concentration of anionic surfactant, sodium dodecyl sulphate in the presence of RF shows that ionic character of RF also contributes to binding and is not solubilized inside the micelle. Significant decrease in concentration of free RF has been observed in the presence of paracetamol. The CD spectrum shows the binding of RF leads to a change in the α helical structure of BSA.

 

Keywords: Riboflavin, bovine serum albumin, fluorescent hydrophobic probe, fluorometric method, circular dichroism studies.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 179-183

 

Rationalization of physico-chemical properties of 5, 6-diarylthiazolo [3,2-b]-1, 2, 4-triazoles towards cyclooxygenase-2(COX-2) inhibition: A QSAR approach

E Manivannan, S Prasanna and S C Chaturvedi*

 

Quantitative structure-activity relationship (QSAR) analysis was performed on a series of 5, 6-diarylthiazolo [3, 2-b]-1, 2, 4-triazoles to explore their possible interaction with the active amino acid residues of cyclooxygenase-2 (COX-2) enzyme. The significance of orientation and conformational rigidity of 5, 6-diarylthiazolo [3, 2-b]-1, 2, 4-triazoles for COX-2 inhibition is discerned by the spatial descriptor principle moment of inertia-X component, PMI-X. The negative contribution of PMI-X indicates the necessity of orientation of substituents towards X-axis of aromatic ring for better activity. The most common electronic interaction between the title compounds and active residues of COX-2 enzyme is corroborated well by the positive contribution of molecular dipole. The contribution of molecular dipole suggests the non-covalent, electronic interactions between 5, 6-diarylthiazolo [3, 2-b]-1, 2, 4-triazoles and binding site of COX-2 enzyme. Our findings reveal the necessity of less bulkier, less polar substituents on the parent structure for better COX-2 inhibitory activity. The limited tolerance of COX-2 enzyme active site towards the bulk of interacting molecules is evident from the negative coefficient of calculated molar refractivity (CMR) in our models.

 

Keywords:    Cyclooxygenase-2 (COX-2) enzyme, non-steroidal anti-inflammatory drugs, quantitative structure-activity relationship, 5, 6-diarylthiazolo [3, 2-b]-1, 2, 4-triazoles, principle moment of inertia-X component (PMI-X), calculated molar refractivity, dipole.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 184-187

 

A study of aromatic hydrogen bonds of peptides with aromatic amino acid side-chains

A Nallini, K Saraboji and M N Ponnuswamy*

 

The importance of hydrogen bonding studies lies in their structural, biological and medicinal applications. Non-conventional hydrogen bonds are weak, but are found to play an important role in biological molecules. In view of their importance a study of the aromatic hydrogen bonds in peptides with aromatic, amino acid side chains was carried out. The results indicate a reasonable probability for their occurrence, thereby enumerating their distinct features.

 

Keywords: Aromatic amino acids; hydrogen bonding; C-H·p interactions

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 41, August 2004, pp. 188-190

 

Effect of aspartame on circadian oscillations of calcium and inorganic phosphorus in rats

P Rajasekar, T Manivasagam and P subramanian*

 

The effect of aspartame on circadian rhythms of calcium and inorganic phosphorus levels was studied in rats. Acrophase delays in calcium rhythms and advances in inorganic phosphorus rhythms and alteration in mesor values in both rhythms were observed in aspartame-treated rats. However, no change in amplitude values was observed. Oral administration of aspartame leads to increased levels of aspartate in the brain, which could alter the characteristics of calcium and inorganic phosphorus rhythms, possibly by modulating transmission in several areas/nuclei in brain, including retinohypothalamic tract (RHT) and suprachiasmatic nuclei (SCN).

 

Keywords: Aspartame, circadian rhythm, calcium, inorganic phosphorus

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, August 2004, pp. 191-194

 

Protein profiles of two rice varieties by 2-D gel electrophoresis under moisture stress

Arti Chandra and Aruna Tyagi*

 

Two rice varieties N22 (drought-tolerant) and Panidhan (drought-susceptible) were studied for the protein profiles under control, moisture stress and abscisic acid (ABA) treatments. Both qualitative and quantitative differences were observed. In control and ABA treatment, the number of polypeptides was more in N22, compared to Panidhan. However, a significant number of polypeptides were induced in Panidhan, compared to N22 under mild and severe stress.

 

Keywords: 2-D Protein profile, moisture stress, rice

 

Author Index

 

Ahmad R

148

Jabeen M

148

Ok Min

141

Ali A M

167

Janave M T

154

 

 

An Sun-Young

141

Jang Moon-Sun

141

Pardhasaradhi B V V

167

Arif S H

148

 

 

Ponnuswamy M N

184

 

 

Kamat B P

173

Prasanna S

179

Basu A

162

Khan M M

148

 

 

Bhattacharyya D

162

Khar A

167

Rajasekar P

188

 

 

Kim Cheorl-Ho

141

Reddy M

167

Chandra A

191

Kim Ji-Youn

141

 

 

Chatterjee M

162

Kumari A L

167

Saraboji K

184

Chaturvedi S C

179

 

 

Seetharamappa J

173

Cho Young-Su

141

Lee Young-Choon

141

Sen P C

162

Choi Yong-Lark

141

 

 

Sharma A

154

 

 

Manivannan E

179

subramanian P

188

Ghosh S

162

Manivasagam T

188

 

 

 

 

Melwanki M B

173

Tyagi A

191

Hasnain A

148

 

 

 

 

 

 

Nallini A

184