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Indian Journal of Biochemistry & Biophysics

 

VOLUME 39

NUMBER 2

APRIL 2002

 
CONTENTS

 

Minireview

 

Unusual catalytic activities and functions of cholinesterases

77

A S Balasubramanian

 

Papers

 

Over-expression of parathion hydrolase of Flavobacterium balustinum in E. coli: Purification and characterization of His-tagged parathion hydrolase

82

Sita Somara, Bramanandam Manavathi, Christoph C Tebbe and

Dayananda Siddavatam*

 

 

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: A 3.2 ns molecular dynamics simulation study

87

Sheeja V Vasudevan and Petety V Balaji*

 

Dipole moment analysis of membrane proteins suggests role in orientation in the membrane

93

G Vasanthi and S Krishnaswamy*

 

 

Hydrogen ions in microaqueous phase during lipase catalysed esterification in non-aqueous media

101

K R Kiran, N G Karanth and S Divakar*

 

 

Interaction of sanguinarine with double stranded RNA structures

106

A Sen and M Maiti*

 

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

113

Nizamuddin Ahmed* and Naganath Swamy

 

Effect of harmaline on intestinal brush border sucrase activity

119

Navneet Kaur, Jyotdeep Kaur and Akhtar Mahmood*

 

Oxidative burden and antioxidant defense system in polymorphonuclear leukocytes of human lung diseases

124

Rashmi N Sharma, A Bhardwaj, D Behera and K L Khanduja*

 

Role of mouse spleen cell HMG proteins and their poly-ADP-ribosylation in betelnut carcinogenesis

130

Theisara Pariat and R N Sharan*

 

 

*Author for correspondence

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 77- 81

 

Unusual catalytic activities and functions of cholinesterases

 

A S Balasubramanian

Received 1 February 2002

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) have been shown to exhibit not only esterase activity but also an amine sensitive aryl acylamidase and a metallo-carboxypeptidase activities. There is also evidence to indicate that they have functions in the substantia nigra of brain, in neural cell differentiation, cell division and tumorigenesis, cell-adhesion and detoxication mechanisms. Butyrylcholinesterase is suggested to act as a back-up enzyme in acetylcholinesterase knock-out mice. Cholinesterases have catalytic or non-catalytic roles in these functions. Partial sequence homology to many other proteins having different functions and a metal binding site which can influence functions are probably factors that confer the non-cholinergic functions and activities on cholinesterases.

 


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 82- 86

 

 

Over-expression of parathion hydrolase of Flavobacterium balustinum in E. coli: Purification and characterization of His-tagged parathion hydrolase

Sita Somara+, Bramanandam Manavathi+, Christoph C Tebbe# and Dayananda Siddavatam*+

Received 9 October 2001; revised and accepted 11 January 2002

The organophosphorus pesticide degrading (opd) gene was cloned downstream to the transcriptional and translational signals of expression vectors pTrc99A and pET32A. The resulting recombinant expression plasmids pNH2 and pHH2 were introduced into E. coli JM105 and E. coli BL21 respectively. On induction the E. coli cells containing pNH2 produced high levels of parathion hydrolase. A 60 kD fusion protein was produced in E. coli cultures containing recombinant plasmid pHH2. The molecular mass of the fusion protein coincided with the molecular mass of 40 kD parathion hydrolase and 20 kD N‑terminal His tag encoded by the vector. Further, the fusion protein was purified using Ni‑column and the N-terminal His-tag was removed by digesting it with thrombin. The resulting protein folded properly in presence of Zn2+ ions, and showed parathion hydrolase activity.


 

 

 

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 87-92

 

 

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: A 3.2 ns molecular dynamics simulation study

 

Sheeja V Vasudevan and Petety V Balaji*

 

Received 10 September 2001; revised and accepted 26 December 2001

 

A dodecylphosphocholine micelle of 86 monomers with 5776 water molecules has been simulated under NPT conditions for 3.2 ns using GROMACS2.0. The micelle was found to be very dynamic. Some of the C-C bonds, independent of their position in the DPC monomer, adopt gauche conformation and the trans gauche transitions are quite frequent. An average of about 11% of the C-C bonds in the micelle are observed to be in the gauche conformation (i.e., |dihedral angle |< 120). The terminal methyl groups are randomly distributed all over the micelle whereas the nitrogen atom of phosphocholine headgroup atoms is restricted to the interface region. Some of the monomers were found to lie on the surface. The shape of micelle, influenced by the packing considerations, shows deviations from spherical shape. The phosphocholine headgroup is well solvated and there is no water penetration into the micelle core. The overall features of the micelle of 86 DPC monomers conforms to the lattice model of micelle proposed by Dill and Flory [Dill K A, Flory P J (1981) Proc Natl Acad Sci USA 78, 676-680] and is similar to DPC micelles of smaller aggregate sizes except for the positional preference of the C-C bonds for the gauche conformation and the trans↔gauche transition times [Tieleman D P, van der Spoel D, Berendsen H J C (2000) J Phys Chem B 104, 6380-6388; Wymore T, Gao X F, Wong T C (1999) J Mol Struct (Theochem) 485-486, 195-210]. It appears that packing considerations play a predominant role in determining the shape and dynamics of the micelle.


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 93-100

 

 

Dipole moment analysis of membrane proteins suggests role

in orientation in the membrane

G Vasanthi and S Krishnaswamy*

Received 29 June 2001; revised and accepted 31 December 2001

The position independent dipole membrane proteins need to be oriented in the membrane in order to function as channels, transporters or recognition systems. Membrane proteins can be broadly classified as either predominantly alpha helical or beta barrel in nature. All the different types of thirteen beta barrel membrane proteins (2OMF, 2POR, 1PRN, 1PHO, 1IIV, 1AF6, 1AOT, 2MPR, 1OSM, 1QJ8, 1BXW, 2FCP and 1FEP) and six alpha helical membrane proteins (1BL8, 1MSL, 1QLB, 1AR1, 1PSS and 1QHJ) from the Protein Data Bank were analyzed. Dipole moment was calculated for both classes of proteins. In all the oligomers, the orientation of the dipole was found to be parallel to direction of insertion that is perpendicular to the possible membrane layer. Monomers do not show a similar orientation. In all the alpha helical oligomers, the dipole points from the intra-cellular to the extra-cellular side. In the oligomeric beta barrel proteins, the direction of the dipole is from the extra-cellular to the intra-cellular side, except for OmpF from E.coli, Omp36 from Klebsiella pneumonia and LamB from E.coli where the situation is reversed. However, the dipole moments of the monomeric proteins and the monomers of the oligomers themselves are not oriented parallel to the molecular axis and the insertion orientation, but they are almost parallel to the membrane surface. It is possible that the quaternary oligomeric association is necessary for the correct orientation in the membrane and this is aided by the dipole orientation. The electrostatic potential surface calculated with all atoms, which also do not show clear separation of charge surfaces. Calculations suggest that backbone structure and oligomer are sufficient for providing the dipole orientation.

 


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 101- 105

 

 

 

Hydrogen ions in microaqueous phase during lipase catalysed
esterification in non-aqueous media

 

K R Kiran, N G Karanth and S Divakar*

Received 21 May 2001; revised 16 October 2001; accepted 12 December 2001

Lipase catalysed stearoyl lactic acid preparation in non-aqueous media was treated as a model system to study the microaqueous phase containing hydrogen ions arising from dissociation of water soluble lactic acid in it. The thermodynamic factors operating at the microaqueous enzyme-water-solvent phase on the lipase in non-polar solvents were investigated in terms of the water of reaction which constitutes the microaqueous phase, partitioning of acid between water of the microaqueous phase and the organic solvent, dissolution and dissociation of the acid and the resultant number of H+ present in the microaqueous phase and the extent of esterification for a given amount of enzyme at various substrate concentrations. Using mass transfer equations, the theoretical number of H+ at the microaqueous phase were calculated and expressed as hydrogen ion numbers to generate plots which indicated various thermodynamic processes operating at the microaqueous phase to maintain this concentration to a safe minimum.

 


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 106- 112

 

 

 

Interaction of sanguinarine with double stranded RNA structures

 

A Sen and M Maiti*

Received 27 May 2001; revised and accepted 21 December 2001

Interaction of sanguinarine with A-form RNA structures of poly(rI)poly(rC) and poly(rA).poly(rU) has been studied by spectrophotometric, spectrofluorimetric, UV melting profiles, circular dichroism and viscometric analysis. The binding of sanguinarine to A-form duplex RNA structures is characterised by the typical bathochromic and hypochromic effects in the absorption spectrum, increasing steady state fluorescence intensity, an increase in fluorescence quantum yield of sanguinarine, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, an increase in the contour length of sonicated rod-like RNA structure and perturbation in circular dichroic spectrum. Scatchard analysis indicates that sanguinarine binds to each polymer in a non-cooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a higher binding affinity of sanguinarine to poly(rI).poly(rC) structure than to poly(rA).poly(rU) structure. On the basis of these observations, it is concluded that the alkaloid binds to both the RNA structures by a mechanism of intercalation.


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 113- 118

 

 

Characterization of a 59 kDa gelatin-binding fragment of
buffalo plasma fibronectin

Nizamuddin Ahmed* and Naganath Swamy

Received 12 February 2001; revised and accepted 14 June 2001

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as : 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 119- 123

 

Effect of harmaline on rat intestinal brush border sucrase activity

Navneet Kaur1, Jyotdeep Kaur1 and Akhtar Mahmood 2*

Received 11 June 2001; revised 11 September 2001

The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing Km by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.

 


 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 124- 129

 

Oxidative burden and antioxidant defense system in polymorphonuclear leukocytes of human lung diseases

Rashmi N Sharma, A Bhardwaj, D Behera$ and K L Khanduja*

Received 4 May 2001; revised and accepted 12 October 2001

Superoxide anion (O2.-) and hydrogen peroxide (H2O2) production was significantly higher in blood neutrophils (PMNs) of patients with lung cancer and non-malignant lung diseases when compared to the controls (p<0.001). Superoxide dismutase (SOD) activity was significantly decreased in PMNs of patients with lung cancer (p<0.001). Similarly, catalase and glutathione peroxidase (GPx) activities were lower in PMNs of lung cancer patients as compared to non-malignant lung diseases and controls. There was an increase in HMP shunt activity as measured by rate of formation of 14CO2 from [1-14C]-glucose in PMNs of lung cancer patients. Modifications in the antioxidant defense system in PMNs of malignant and non-malignant lung diseases, the changes being more in the malignancy are indicated.

 

Indian Journal of Biochemistry & Biophysics

Vol. 39, April 2002, pp. 130- 132

 

 

 

Role of mouse spleen cell HMG proteins and their poly-ADP-ribosylation

in betelnut induced carcinogenesis

 

Theisara Pariat and R N Sharan*

 

Received 23 April 2001; revised 27 September 2001; accepted 18 October 2001

The role of high mobility group (HMG) proteins and their poly-ADP-ribosylation (PAR) in betel nut induced initiation of carcinogenesis in mice has been studied. A known carcinogen, diethylnitrosamine (DEN) was used as a positive control. Swiss albino mice were chronically exposed to aqueous extract of betel nut (AEBN) or DEN at low doses for up to 4 weeks. The poly-ADP-ribosylation (PAR) of spleen cell HMG proteins was monitored using [32P]-NAD+. Parallel to this, chromatin was subjected to DNase I cleavage and the organizational state of the chromatin was monitored. The PAR of HMG proteins showed a marked progressive reduction at different times following AEBN- or DEN treatment. HMG proteins isolated from the control and carcinogen treated mice were allowed to reassociate with the untreated spleen cells chromatin. The reassociated chromatin showed progressive relaxation in its superstructure. The results suggest that under the influence of potential carcinogens AEBN or DEN, the mouse spleen cell HMG proteins created molecular conditions favourable to initiation of cancer.