Volume 6, Issue 1
March 1984, pages 1-153
pp 1-16 March 1984
Conformations of valinomycin and its complexes with Perchlorate and thiocyanate salts of barium, in a medium polar solvent acetonitrile, were studied using nuclear magnetic resonance spectroscopic techniques. Valinomycin was shown to have a bracelet conformation in acetonitrile. With the doubly charged barium ion, the molecule, at lower concentrations, predominantly formed a 1:1 complex. At higher concentrations, however, apart from the 1:1, peptide as well as ion sandwich complexes were formed in addition to a ‘final complex’. Unlike the standard 1:1 potassium complex, where the ion was centrally located in a bracelet conformation, the 1:1 barium complex contained the barium ion at the periphery. The ‘final complex’ appeared to be an open conformation with no internal hydrogen bonds and has two bound barium ions. This complex was probably made of average of many closely related conformations that were exchanging very fast (on nuclear magnetic resonance time scale) among them. The conformation of the ‘final complex’ resembled the conformation obtained in the solid state. Unlike the Perchlorate anion, the thiocyanate anion seemed to have a definite role in stabilising the various complexes. While the conformation of the 1:1 complex indicated a mechanism of ion capture at the membrane interface, the sandwich complexes might explain the transport process by a relay mechanism.
pp 17-35 March 1984
The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 188.8.131.52) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4+, and further, the enzyme is repressed by increasing concentrations of NH4+. In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH4+ or
L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.
Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M-1 min-1 and 760 M-1 min-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.
pp 37-46 March 1984
Relative α-lipoic acid content of diabetic livers was considerably less than that of normal livers as determined by gas chromatography. It was not possible to detect any dihydrolipoic acid in the livers. Biochemical abnormalities such as hyperglycaemia, ketonemia, reduction in liver glycogen and impaired incorporation of [2-14C] -acetate into fatty acids in alloxan diabetic rats were brought to near normal levels by the oral or intraperitoneal administration of dihydrolipoic acid. The effect of α-lipoic acid was comparable to that of dihydrolipoic acid in reducing the blood sugar levels of diabetic rabbits during a glucose tolerance test.
The results suggest that the mode of action of lipoic acid was through stimulation of pyruvate dehydrogenase.
pp 47-59 March 1984
The development of α-amylase (EC 184.108.40.206) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (α-amylase I and α-amylase II), with different molecular weights and isoelectric points. α-Amylase I contained a much higher content of sugars than α-amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in α-amylase I and the rest in a-amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. α-Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas α-amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components.
pp 61-67 March 1984
Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress.
pp 69-77 March 1984
Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)2SO4. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg2+, Co2+, Ca2+ or Mn2+. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co2+)-dependent enzyme. The apparentKm values of the enzyme for casein, ATP and Co2+ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine. The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized.
pp 79-85 March 1984
The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.
pp 87-95 March 1984
Genetic organization of a proximal region of the second chromosome inDrosophila melanogaster has been analysed by saturation mutagenesis. Seven alleles were uncovered in this region in addition to the one previously known. Besides this, quite a few mutations were isolated that non-complemented more than one group of lethals and looked very much like deletions of varying extent. Except one, all the lethals complemented M(2)z.
pp 97-105 March 1984
The fatty acid composition of thoracic and abdominal aortic intima and media of normal human subjects and rhesus monkeys has been studied. Significantly higher values of unsaturated fatty acids as compared to saturated fatty acids have been noted in the intima of monkey as compared to man. The fatty acid profile of the aortic wall in these two species has provided a probable biochemical basis for the lesser incidence of atherosclerosis in macaques.
pp 107-113 March 1984
Aortal collagen typing in monkey and man showed the presence of types I, HI and V in human aorta and types I and III in monkey aorta. Type III collagen was found to be a predominate type in both species. The molecular weight of type III collagen was similar in these species while type I collagen was different. Both monkey and human collagen types I and III were found to be immunogenic. Type I collagen was significantly increased while type III was decreased in human atherosclerotic plaque. Collagen typing in fatty streak remained unaltered.
pp 115-123 March 1984
Mungbean contains three isoenzymes of superoxide dismutase designated isoenzyme I, II and III. The two cytosolic superoxide dismutases (I and II) were purified to homogeneity by ammonium sulphate fractionation, ion exchange chromatography on diethylaminoethyl cellulose, gel filtration and preparative polyacrylamide.gel electrophoresis. The molecular weights of isoenzyme I and isoenzyme II were determined to be 33,000 and 31,600 respectively. The subunit molecular weight was approximately 16,000 indicating that the isoenzymes contained two identical subunits. The ultra-violet absorption spectra revealed a maximum at 258–264 nm for the two isoenzymes. Superoxide dismutase I and II were inhibited to different extents by metal chelators. Isoenzyme I was more sensitive to inhibition by cyanide and azide, while isoenzyme II was more susceptible to inhibition by diethyldithiocarbamate ando-phenanthroline. Both the isoenzymes exhibited similar denaturation profiles with heat, guanidinium chloride and urea. The denaturation with urea and guanidinium chloride was reversible. The two copper-zinc enzymes were more stable towards thermal inactivation compared to manganese and iron superoxide dismutases from other sources. The results indicate that the two isoenzymes differ from each other only with respect to charge and sensitivity towards metal chelators.
pp 125-134 March 1984
Reduction inFc receptor expression as assayed by ‘erythrocyte’ rosetting of macrophage cultures from long term treated lepromatous leprosy patients (bactereologically negative) was seen in the presence of viableMycobacterium leprae. Macrophages with and without intracellular bacilli demonstrated this reduction. On the basis of this observation the conditioned medium ofMycobacterium leprae infected macrophage cultures of lepromatous patients, were tested on macrophages from normal individuals for [3H]-leucine incorporation and antigen specific physical interaction with lymphocytes. Both these parameters showed decreased values as compared to the controls which were not exposed to this conditioned medium. Lymphocyte transformation toMycobacterium leprae in leucocyte cultures of normal individuals was also reduced in the presence of the conditioned medium from lepromatous patients’ macrophages. The indication that this factor may be a prostaglandin was suggested by the observation that its synthesis was inhibited by indomethacin. Its importance in the non-specific depression in cell-mediated immunity seen in lepromatous patients is discussed.
pp 135-145 March 1984
A simple apparatus for vertical.,in situ, polyacrylamide or agarose gel casting as well as for the subsequent electrophoresis is described. The apparatus is completely leakproof and does not require any special device like clamps, O-rings, gaskets, grease etc. for sealing. Slab gels of various thickness (0.04 to 1.0 cm) can be made and the apparatus can be used for analytical or preparative purposes. Gel rods can also be cast and run in the device. Forward as well as reverse polarity electrophoresis of a sample can be run simultaneously in the apparatus.
pp 147-153 March 1984
Male and female albino rats (Wistar strain) were given single and multiple doses of karaya gum suspended either in peanut oil or mixed with basal diet at different concentrations ranging from 0.5 to 8 g gum/kg body weight. The plant gum did not elicit any overt signs of toxicity or death in both sexes of rats.
Daily administration of karaya gum mixed with basal diet at different dose levels (0, 5, 20 and 40 g gum/kg diet) for a period of 90 days showed no adverse effects in male and female rats. The body weight, growth pattern, food and water intake were comparable with those of the normal rats. There were no significant biochemical, or morphological alterations in the vital organs of experimental animals.