Indian Journal of Biochemistry & Biophysics

Contents

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 77-91

Diversity of calcium action in regulation of mammalian calmodulin-dependent cyclic nucleotide phosphodiesterase

Rajendra K Sharma

Department of Pathology, College of Medicine, University of Saskatchewan, Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, SK Canada S7N 4H4

Received 11 November 2002; revised 5 February 2003

Calmodulin(CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) plays a critical role in the complex interactions between the cyclic nucleotide and Ca2+ second messenger systems. Bovine brain contains two major PDE1 isozymes, designated according to tissue origin and subunit molecular mass as brain 60 kDa and 63 kDa PDE1 isozymes. Kinetic properties suggest that 63 kDa PDE1 isozyme is distinct from 60 kDa, heart and lung PDE1 isozymes. Although 60 kDa, heart and lung PDE1 isozymes are almost identical in immunological properties, they are differentially activated by calmodulin (CaM). These isozymes are further distinguished by the effects of pharmacological agents. Another main difference is that 60 kDa PDE1 isozyme is a substrate of cAMP-dependent protein kinase, whereas, 63 kDa PDE1 isozyme is phosphorylated by CaM-dependent protein kinase. The phosphorylation of PDE1 isozymes is accompanied by a decrease in the isozyme affinity towards CaM, and it can be reversed by a CaM-dependent phosphatase (calcineurin). The complex regulatory properties of PDE1 isozymes are precisely regulated by cross-talk between the Ca2+ and cAMP signaling pathways.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 92-97

Identification of serotonin-sensitive aryl acylamidase activity with

cobra venom acetylcholinesterase

Ramanna V Rajesh, Aiylam S Balasubramanian and Rathanam Boopathy*

Department of Biotechnology, Enzyme Biotech Lab, Bharathiar University, Coimbatore 641 046, India

Received 13 May 2002; revised 11 October 2002

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40°C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 108-113

Site-specific mutations localized in the D-E loop of the D1 protein of photosystem II affect phototolerance in Synechocystis sp.

PCC 6803 containing psbAII gene

Munna Singh1* and Kimiyuki Satoh2

1Department of Plant Physiology, CBSH, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, India

2Department of Biology, Okayama University, Okayama 700, Japan

Received 18 July 2002; revised 6 February 2003

Photosynthetic characteristics along with phototolerance and photoinhibition of photosystem II (PS II) were monitored in Synechocystis sp. PCC 6803 wild type (KC) and its psbAII mutants viz., I6 (N322I, I326F, and F328S), G6 (N267Y), and H7 (Y254C and I314V) that have up to three point mutations, localized in the D-E loop of the D1 polypeptide of PSII reaction centre. These strains exhibited entirely different growth trends upon shifting from 30 mmol m-2s-1 to high irradiance (500 mmol m-2s-1, 30°C). The I6 and H7 cells grew well, whereas KC and G6 cells showed inability for cell multiplication. The photosynthetic efficiency demonstrated about 50% loss in chlorophyll fluorescence of variable yield (Fv/Fm) within 20-30 min in all mutants, whereas the wild type (KC) cells could reach the same level of loss in 2 hr. I6 and H7 cells showed continuous cell growth and maintenance under long-term exposure of high light compared to G6 mutant and wild type cells. The wild type cells showed slow decrease in their photochemical activity and Fv/Fm values, compared to mutant cells. The recovery seemed to be almost identical, and also stimulated by growth light, inspite of differential photoinhibitory behaviours. Darkness and translational inhibitor lincomycin both were found to be unassociated with the restoration of photoinhibited process of PS II.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 114-121

Structural alterations of phospholipid film domain morphology induced by cholesterol

A K Panda1,2*, A Hume2, K Nag2,3, R R Harbottle2 and N O Petersen2*

1Department of Chemistry, Behala College, Kolkata 700 060, India

2Department of Chemistry, The University of Western Ontario, London, ON-N6A 5B7,Canada

3Dept. of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland A1B 3X9, Canada

Received 23 July 2002; revised 31 January 2003

Structures of the monolayer films of dipalmitoylphosphatidylcholine (DPPC) mixed with different amounts of cholesterol were studied at air-water interface using surface pressure-area measurements, epifluorescence microscopy and atomic force microscopy (AFM). Pure DPPC, cholesterol or DPPC-cholesterol mixtures were dissolved in organic solvents with a small amount of fluorescently labeled phospholipid probe (NBD-PC) and spread onto the air-water interface. Surface pressure-area isotherms and epifluorescence microscopy of such films at the air-water interface suggested that DPPC undergoes a gas to fluid to condensed phase transition, while cholesterol undergoes a gas to solid-like transition. A shift of the surface pressure-area curve to lower area per molecule was observed when cholesterol was mixed with DPPC. Epifluorescence microscopy showed the formation of spiral shaped domains for mixed monolayers. Increase in cholesterol content abolished domain characteristics possibly due to fluidizing property of cholesterol. AFM measurements of monolayers, transferred onto freshly cleaved mica by Langmuir-Blodgett technique, revealed the alterations caused by cholesterol on the gel and fluid domains of such films. AFM measurements re-established similar trend in domain characteristics as evidenced in epifluorescence microscopy.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 122-130

Interaction of water vapour with twenty different poly-l-amino acids and their excess hydration in presence of sodium chloride

N Ghosh, P Dutta, P Mahapatra, K P Das# and D K Chattoraj*

Department of Food Technology & Biochemical Engineering, Jadavpur University, Kolkata 700 032

#Bose Institute, 93 Acharya Prafulla Chandra Road, Kolkata 700 009

Received 16 May 2002; revised 13 January 2003; accepted 3 March 2003

Using the isopiestic vapour pressure technique, the magnitudes of excess binding of water and NaCl per mole of twenty different poly-l-amino acid residues, respectively in the presence of different bulk molefractions (X2) of NaCl have been evaluated from the mathematical expressions for the Gibbs surface excesses. At certain high ranges of NaCl concentration, the plot of  vs. X1/X2 becomes linear, so that moles of water and NaCl, respectively bound per mole of amino acid residue can be evaluated.  is the excess moles of H2O per mole of amino acid residue and X1 and X2 stand for mole fractions of the water and NaCl, respectively in the sample system. Also, using the integrated form of the Gibbs absorption equation, the values of standard free energy change for the excess adsorption of NaCl per kg of poly-l-amino acids have been evaluated. These values are all positive as a result of positive excess hydration of polyamino acids. The standard free energy of excess hydration  (equal to ) is negative due to spontaneous excess hydration of polyamino acid in the presence of a salt.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 131-135

Adverse health effects in workers exposed to trace/toxic metals at workplace

Rita Mehra* and Meenu Juneja

Department of Pure and Applied Chemistry, Maharshi Dayanand Saraswati University, Ajmer 305 009, India

Received 16 July 2002; revised 23 October 2002

Widespread use of metals in industrial activities has enhanced the occupational exposure to toxic metals as well as the health risks of metal hazards to humans. Elemental analysis in human tissues is the most common application of biological monitoring for screening, diagnosis and assessment of such exposures and risk. Among various biopsy materials, blood, hair, nail, teeth and body fluids may be used as bioindicators for this purpose. The present paper deals with the determination of Pb, Cr, Ni, Mn, Fe, Cu and Zn elemental concentration in workers exposed to these metals at workplace by atomic absorption spectrophotometry, with adequate quality control measures using hair as biopsy material. The study group includes the male workers such as welders, foundry man, fitter, hammer man, machine man, cupola man etc., besides office workers of locomotive workshop in Ajmer and surrounding areas exposed to different metals. Age and sex matched controls of persons working in the same area of work in offices etc. and not exposed to metal pollution were selected for valid comparison. It is proposed to validate the use of hair as a biological marker for assessing metal body burden of workers. In our study significant correlations have been found between skin disease and Cr, Mn, Fe, Cu; chest pain and Pb; hypertension and Cu, Mn; mental stress and Mn, Ni, Cu, Zn; liver problem and Ni; indigestion and Cr; Ni, diabetes and Cr, Mn, Ni; tuberculosis and Zn; breathing trouble and Cr, Mn, Fe, Ni, Zn. The advantages of choosing hair as a biopsy material are also given.

Indian Journal of Biochemistry & Biophysics

Vol 40, April 2003, pp. 136-138

Liver and colon pro- and anti-oxidant enzyme activities in rats after long-term ethylnitrosourea exposure

Yavuz Silig1* Ozturk Ozdemir2 and Atilla Atalay3

1Department of Chemistry, Faculty of Sciences and Arts,

2Department of Medical Biology and  3Genetics and Biochemistry Faculty of Medicine, Cumhuriyet University,

58140-Sivas, Turkey

Received 30 July 2002; revised 28 February 2003

Liver and colon pro- and anti-oxidant enzyme activities were investigated in rats treated with ethylnitrosourea (ENU) (i.p.) (4 mg/kg body wt) for 6 months. The pro-oxidant enzymes (NADPH cytochrome c reductase, NADH cytochrome c reductase, NADH cytochrome b5 reductase and cytochrome P-4502E1 and the anti-oxidant enzyme, superoxide dismutase (SOD) exhibited significantly increased activity in liver and colon. Glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase (GST) showed enhanced activity in liver, but decreased activity in colon. Glutathione peroxidase (GP) and glutathione reductase (GR) activities were significantly increased in colon, but decreased in liver. Catalase (CAT) activity while showed a significant increase in liver, exhibited only marginal increase in colon. Malondialdehyde (MDA) level was significantly elevated in both tissues.

Indian Journal of Biochemistry & Biophysics

 Vol 40, April 2003, pp. 139-

Effect of gastrointestinal proteases on purified human intrinsic factor-vitamin B12 (IF-B12) complex

K Srikumar1* and R Premalatha2

 1Dept. of Biological Sciences and 2Community College, Pondicherry University, Kalapet, Pondicherry 605014, India

Received 7 July 2002; revised 26 February 2003

Intrinsic factor (IF) from human gastric juice was purified and complexed with vitamin B12 (IF-B12 complex) on Sepharose-vitamin B12 affinity matrix. By labeling studies, using [57Co] vitamin B12 and 125I, the specific B12 binding activity of IF was found to be 23 mgB12/mg protein, and the molecular size by gel filtration 60 kDa. Proteolysis of the IF-B12 complex by sequential treatment with pepsin, trypsin, α-chymotrypsin and carboxypeptidase A, followed by chromatography of proteolysed complex and IF-B12 showed higher mobility of proteolysed fraction. Gel filtration, however, showed same molecular size for both proteolysed and the IF-B12 complex. On SDS-PAGE, purified IF-B12 appeared as a single band of 60 kDa. The proteolysed complex had higher mobility on SDS-PAGE and did not bind to zirconium phosphate gel. Immunodiffusion with rabbit antisera had positive reaction with IF-B12, but there was no reaction with the proteolysed sample.