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Indian Journal of Biochemistry & Biophysics

 

VOLUME 39

NUMBER 3

JUNE 2002

 
CONTENTS

 

Minireview

 

Neurotransmission by ATP: New insights, novel mechanisms

137

  Parakashtha Ghildyal and Rohit Manchanda*

 

 

 

Papers

 

Enhanced expression of heterotrimeric GTP-binding protein subunits in Zajdela ascitic hepatoma

     148

  S K Sharma and M R Das*

 

 

 

Effect of urea at lower concentration on the structure of papain ¾ Formation of a stable molten globule and its characterization

155

  H A Sathish, P Ramesh Kumar and V Prakash*

 

 

 

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: Uptake and assimilation of inorganic-N and amino acids

163

  Jyotirmoy Bhattacharya, Arvind Kumar Singh and Amar Nath Rai*

 

 

 

Studies on the microstructure of bile salt aggregates in aqueous n-alkanol solutions by Small Angle Neutron Scattering

170

        J Santhanalakshmi*, G Shanthalakshmi, V K Aswal and P S Goyal

 

 

 

Purification, characterization and amplification of a 1.8 kbp fragment of xylanase 5 from Aeromonas caviae W-61

179

  Narayan Roy* and Yoshiyuki Kamio

 

 

 

Expression of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine

185

  S Mahmood*, C P Sodhi and N K Ganguly

 

 

 

A spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2: Comparison with fluorometric assay

191

  Shipra Rastogi, Subhash K Khanna and Mukul Das*

 

 

 

Cardioprotective effect of coconut kernel protein in isoproterenol administered rats

197

  S Mini and T Rajamohan*

 

 

——————

*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.