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Indian Journal of Biochemistry & Biophysics
VOLUME 39
NUMBER 3
JUNE 2002
CONTENTS
Minireview
Neurotransmission by ATP: New insights, novel mechanisms
Parakashtha Ghildyal and Rohit Manchanda*
137
Papers
Enhanced expression of heterotrimeric GTP-binding protein subunits in Zajdela
ascitic hepatoma
S K Sharma and M R Das*
148
Effect of urea at lower concentration on the structure of papain  Formation of a
stable molten globule and its characterization
H A Sathish, P Ramesh Kumar and V Prakash*
155
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*
163
Studies on the microstructure of bile salt aggregates in aqueous n-alkanol solutions
by Small Angle Neutron Scattering
J Santhanalakshmi*, G Shanthalakshmi, V K Aswal and P S Goyal
70
1
Purification, characterization and amplification of a 1.8 kbp fragment of xylanase 5
179
from Aeromonas caviae W-61
Narayan Roy* and Yoshiyuki Kamio
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
185
A spectrophotometric method to monitor the catalytic activity of microsomal
191
cytochrome P-450 IIB1/2: Comparison with fluorometric assay
Shipra Rastogi, Subhash K Khanna and Mukul Das*
Cardioprotective effect of coconut kernel protein in isoproterenol administered rats
S Mini and T Rajamohan*
197
——————
*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 P 2X
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 (G2) 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 G2 in ZAH is regulated at
transcriptional as well as post-transcriptional levels. Gi3 is also over-expressed in ZAH. No amplification or any
change in the organization of the genes for G2 or Gi3 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
 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 (g3/g2)T,1,3 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 N 2 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-1,4xylanase 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 M, 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 CO 2 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.
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