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address: http://www.bioline.org.br/ib
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Indian
Journal of Biochemistry & Biophysics
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VOLUME 39 |
NUMBER 3 |
JUNE 2002 |
CONTENTS
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Minireview |
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Neurotransmission
by ATP: New insights, novel mechanisms |
137 |
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Papers |
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Enhanced
expression of heterotrimeric GTP-binding protein subunits in Zajdela ascitic
hepatoma |
148 |
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Effect of urea at lower
concentration on the structure of papain ¾ Formation of a stable
molten globule and its characterization |
155 |
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Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: Uptake and assimilation of
inorganic-N and amino acids |
163 |
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Jyotirmoy
Bhattacharya, Arvind Kumar Singh and Amar Nath Rai* |
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Studies on the microstructure of bile salt aggregates in aqueous n-alkanol solutions by Small Angle Neutron Scattering |
170 |
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J
Santhanalakshmi*, G Shanthalakshmi, V K Aswal and P S Goyal |
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Purification, characterization and amplification
of a 1.8 kbp fragment of xylanase 5 from Aeromonas
caviae W-61 |
179 |
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Expression of
sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine |
185 |
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A spectrophotometric method
to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2:
Comparison with fluorometric assay
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191
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Cardioprotective
effect of coconut kernel protein in isoproterenol administered rats |
197 |
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——————
*Author
for correspondence
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 137- 147
Minireview
Neurotransmission By ATP: New Insights, Novel Mechanisms
Parakashtha
Ghildyal & Rohit Manchanda
Received 5 April 2002
Purines have long been known
for their roles in extracellular signaling. One of the most interesting
functions to come to light recently has been the involvement, particularly of
adenosine 5’-triphosphate (ATP), as a neurotransmitter in the central and the
sympathetic nervous system. ATP is
stored in and released from synaptic nerve terminals, like other
neurotransmitters, and is known to act postsynaptically via specific
rapidly-conducting, ligand-gated ion channels, the P2X
receptors. Another interesting feature
is the discovery that ATP is widely found to be a “co-transmitter” at the same
synapses in combination with other neurotransmitters such as noradrenaline,
acetylcholine, and GABA, altering our picture of the biophysics and
biochemistry of neurotransmission at these synapses. We describe here these and other aspects of neurotransmission by
ATP being investigated vigorously today, including recent findings on P2X
receptors and those on the synaptic inactivation of ATP by ecto-ATPase. We conclude by pointing out possible
pharmacological and clinical implications of neurotransmission by ATP.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 148- 154
Enhanced
expression of heterotrimeric GTP-binding protein subunits in
Zajdela
ascitic hepatoma
Sharma
S K and Das M R
Received 31
December 2001; accepted 1 April 2002
G protein beta subunit (Gb2)
is over-expressed in Zajdela ascitic hepatoma (ZAH), a rat ascitic tumor, at
mRNA as well as protein levels. Nuclear run-off transcription analysis suggests
that the expression of Gb2 in ZAH is regulated at
transcriptional as well as post-transcriptional levels. Gai3
is also over-expressed in ZAH. No amplification or any change in the
organization of the genes for Gb2 or Gai3
was observed. It is possible that over-expression of G protein subunits in ZAH
could provide proliferative advantage to the cells by virtue of their effects
on second messenger systems.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 155- 162
Effect of urea
at lower concentration on the structure of
papain ¾ formation of
a stable molten globule and
its characterization
H A Sathish, P
Ramesh Kumar and V Prakash
Received 10
September 2001; revised and accepted 1 April 2002
Effect of lower concentrations of urea
on papain was monitored by optical spectroscopy, calorimetry and partial
specific volume measurements. At lower
concentrations of urea, papain exhibits a different structure and
showed an increase in the intensity of circular dichroic (CD) spectra as
compared to the native molecule. At
lower concentrations (0.2 – 1.5 M) of urea, binding of
8-Anilino-naphthalene sulfonic acid
(ANS) to the papain molecule was higher, at 0.5 M there was about 50%
increase in ANS binding. Both
calorimetric and spectroscopic studies indicated an increased thermal stability
of the molecule at lower concentrations. At 0.5 M urea concentration, the apparent thermal denaturation temperature
increased from a control value of 83 ± 1o C to 86 ± 1o C. At isopotential conditions, the
partial specific volume of papain was found to be higher in presence of lower
concentrations of urea, than the native protein or unfolded molecule. The preferential interaction parameter (dg3/dg2)T,m1,m3 showed a negative value in the presence of
lower concentrations of urea (0.2 – 2 M),
which is maximum at 1 M urea with a
value of – 0.019 g/g. Above 3 M
urea, the preferential interaction parameter was positive.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 163- 169
Nitrogen
nutrition in the cyanobacterium Nostoc
ANTH, a symbiotic isolate from Anthoceros:
Uptake and assimilation of inorganic-N and amino acids
Jyotirmoy
Bhattacharya, Arvind Kumar Singh and Amar Nath Rai
Received 16
November 2001; revised and accepted 20 March 2002
Amino acid uptake and utilization of various
nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and its mutant (Het-Nif-)
isolate defective in heterocyst formation and N2-fixation. Both
parent and its mutant grew at the expense of glutamine, asparagine and arginine
as a source of fixed-nitrogen. Growth was better in glutamine- and
asparagine-media as compared to that in arginine media. Glutamine and asparagine
repressed heterocyst formation, N2-fixation and nitrate reduction in
Nostoc ANTH, but arginine did so only
partially. The poor growth in arginine-medium was not due to poor uptake rates,
since the uptake rates were not significantly different from those for
glutamine or asparagine. The glutamine synthetase activity remained unaffected
during cultivation in media containing any one of the three amino acids tested.
The uptake of amino acids was substrate-inducible, energy-dependent and
required de novo protein synthesis.
Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of
amino acids. In N2-medium (BG-110), the uptake of
ammonium and amino acids in the mutant was significantly higher than its parent
strain. This was apparently due to nitrogen limitation since the mutant was
unable to fix N2 and the growth medium lacked combined-N.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 170- 178
Studies
on the microstructure of bile salt aggregates in aqueous
n-alkanol
solutions by Small Angle Neutron Scattering
J Santhanalakshmi*,1, G Shanthalakshmi2, V K Aswal3 and P S Goyal4
Received 27 September 2001; revised 11 February 2002; accepted 1 March 2002
Small Angle
Neutron Scattering (SANS) measurements on two bile salt micelle systems sodium
cholate (NaC) and sodium deoxycholate (NaDC) each 0.1 M in the absence and presence of 0.05 M 1-butanol, 1-pentanol, 1-hexanol and 1-octanol were carried out
in D2O at ambient conditions (25°C). The scattering cross-section as
a function of wave vector transfer (Q) showed the presence of correlation peaks
characteristic of strong inter micellar interactions. The correlation peak
positions were shifted to low Q values in the presence of n-alkanols for both NaC and NaDC micelles. Best fit curves of SANS
were produced by applying Hayter-Penfold analysis considering monodisperse
ellipsoid model for the micelles. The best fits were found for constant
semi-minor axis=8Å with an increasing semi-major axis for increasing n-alkanol chain lengths. The micellar
growths in presence of n-alkanols
were studied using the ESR correlation time measurements on suitable spin probe
incorporated micelles. The growth parameter and the hydrodynamic radii values
were found to be agreeable with SANS data. Intermicellar interaction potentials
seem to increase with the Cn of n-alkanols.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 179- 184
Purification, characterization and
amplification of a 1.8 kbp fragment of xylanase 5 from Aeromonas caviae
W-61
Narayan Roy and Yoshiyuki
Kamio
Received 23 April 2001; revised and
accepted 19 April 2002
Aeromonas caviae W-61 produces multiple extracellular
xylanases, the xylanases 1, 2, 3, 4, and 5.
In this study, we purified and characterized the xylanase 5 of A.
caviae W-61, and amplified a part of xylanases 5 gene (xyn5). The
purified xylanase 5 was found to be a single polypeptide with molecular mass of
140 kDa. It was an endo-β-1,4-xylanase showing optimum temperature 40ºC,
optimum pH 6.0. Xylobiose, xylotriose, xylotetrose, xylopentose,
xylohexose and a small amount of xylose were detected as the hydrolysis
products. The N-terminal amino acid sequence and several internal amino acid
sequences of xylanases 5 was determined. From the sequence, a 1.8 kbp fragment was amplified by PCR using
forward and reverse primers. DNA sequencing confirmed the presence of
nucleotide sequences corresponding to the N-terminal amino acid sequence and
the internal amino acid sequences of xylanase 5.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 185- 190
Expression
of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine
S Mahmood*, C P Sodhi and N K Ganguly
Received 24 August 2001; revised 13
December 2001
The absorption of D-glucose and brush border
membrane disaccharidases in the intestine of rat during infection by Giardia lamblia has been studied. The
level of mRNA encoding Na+/glucose co-transporter (SGLT1) and brush
border sucrase and lactase activities were also analyzed. At the peak of
infection, i.e, day 7, 11 and 15 post-infection, there was a marked decrease in
the signal of 4.5 kb and 2.8 kb mRNAs encoding SGTL1 compared to the controls.
A similar decrease in sucrase and lactase mRNA’s (6.5 kb and 6.8 kb
respectively) was also observed under these conditions. This corresponds to
observed decrease in the rate of Na+-dependent D-glucose uptake and
low activities of brush border sucrase and lactase under these conditions.
There was no change in Na+-independent D-glucose uptake in giardia
infected rat intestine. These findings suggest that the down regulation of the
expression of SGLT1 and brush border sucrase and lactase activities may be
responsible for the observed malabsorption in G. lamblia infection.
Indian Journal
of Biochemistry & Biophysics
Vol. 39, June 2002, pp. 191- 196
A spectrophotometric method to monitor the catalytic activity of
microsomal cytochrome P-450 IIB1/2: Comparison with fluorometric assay
Shipra Rastogi, Subhash K.
Khanna and Mukul Das
Received 6 June 2001;
revised 18 December 2001; accepted 11 February 2002
A simple spectrophotometric method to monitor the
catalytic activity of microsomal cytochrome P-450 IIB1/2 has been developed.
The method employs measurement of utilization of NADPH, consumption of the
substrate, pentoxyresorufin (PRF) and formation of the product, resorufin (RF)
in the same reaction mixture containing hepatic microsomes from phenobarbital
treated rats. The velocity of NADPH utilization (16..36 nmol/min/nmol P-450),
PRF consumption (1.58 nmole/min/nmole P-450) and RF formation (1.57
nmole/min/nmole P-450) suggested a stoichiometry of 1:1 between the substrate
and the product along with utilization of 10 molecules of NADPH. However, the Km for the enzyme activity
(nmole RF formed/min/nmole P-450) using varying concentrations of PRF and NADPH
as substrates were found to be 11.6 and 20.2 mM,
respectively. The spectrophotometric method was compared with fluorometric
method in terms of linearity with time, P-450 content and Vmax, Km values
observed for the reaction. Inhibition studies with metyrapone and SKF 525A in
the utilization of NADPH, consumption of PRF and formation of RF suggested that
the method could be useful in monitoring the effect of various inhibitors on
the P-450 IIB1/2 reaction.
Indian Journal of
Biochemistry & Biophysics
Vol. 39, June 2002, pp. 197- 200
Cardioprotective effect of coconut kernel protein in
isoproterenol administered rats
S Mini and T Rajamohan*
Received 21
June 2001; revised 11 April 2002
Male albino rats were given subcutaneous injection
of isoproterenol (10 mg/100g body wt) twice at an interval of 24 hr to induce
myocardial infarction. The rats showed massive myocardial necrosis and
increased activities of creatinine phosphokinase (CPK), glutamate oxaloacetate
transaminase (GOT) and glutamate pyruvate transaminase (GPT), in serum, while a
decrease in nitric oxide synthase activity and lower levels of palmitate
oxidation into CO2 and ATP were observed in the heart. Rats
pre-treated with coconut protein or l-arginine
showed significantly decreased CPK, GOT and GPT activities in the serum. There
was significantly higher nitric oxide synthase activity and higher rate of
palmitate oxidation into CO2 and increased levels of ATP in the
heart in these groups. These observations indicate the cardioprotective effect
of coconut protein, which may be attributed to the high content of l-arginine present in it.