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Indian Journal of Biochemistry & Biophysics
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VOLUME 39
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NUMBER 2
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APRIL 2002
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CONTENTS
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*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 |< 120º). 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.
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.
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.
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.
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.