Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

Role of Ca2+ ion on Leishmania-macrophage attachment

Summary Leishmania donovani, the etiological agent for the disease visceral leishmaniasis, attach themselves to the macrophages for initiation of the disease. The attachment process has been found to be regulated by Ca²⁺ ions. Verapamil, a Ca²⁺-channel blocker inhibits Leishmania-macrophage attachment. The inhibitory effect is increased with time. Nifedipine, another Ca²⁺-channel blocker exhibits the same effect. The attachment process is stimulated by Ca²⁺-ionophore alone. The inhibitory effects of the calcium channel blockers are reversed by the ionophore.     

Image segmentation using a neural network

An object extraction problem based on the Gibbs Random Field model is discussed. The Maximum a'posteriori probability (MAP) estimate of a scene based on a noise-corrupted realization is found to be computationally exponential in nature. A neural network, which is a modified version of that of Hopfield, is suggested for solving the problem. A single neuron is assigned to every pixel. Each neuron is supposed to be connected only to all of its nearest neighbours. The energy function of the network is designed in such a way that its minimum value corresponds to the MAP estimate of the scene. The dynamics of the network are described. A possible hardware realization of a neuron is also suggested. The technique is implemented on a set of noisy images and found to be highly robust and immune to noise.     

Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.

Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.     

Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): pathway and properties of enzymes of sn-glycerol 3-phosphate formation

The enzymatic pathway for the synthesis of sn-glycerol 3-phosphate was investigated in developing groundnut seeds (Arachis hypogaea). Glycerol-3-phosphate dehydrogenase was not detected in this tissue but an active glycerokinase was demonstrated in the cytosolic fraction. It showed an optimum pH at 8.6 and positive cooperative interactions with both glycerol and ATP. Triosephosphate isomerase and glyceraldehyde-3-phosphate phosphatase were observed mainly in the cytosolic fraction while an active glyceraldehyde reductase was found mainly in the mitochondrial and microsomal fractions. The glyceraldehyde 3-phosphate phosphatase showed specificity and positive cooperativity with respect to glyceraldehyde 3-phosphate. The glyceraldehyde reductase was active toward glucose and fructose but not toward formaldehyde and showed absolute specificity toward NADPH. It is concluded that in the developing groundnut seed, sn-glycerol 3-phosphate is synthesized essentially by the pathway dihydroxyacetone phosphate----glyceraldehyde 3-phosphate Pi----glyceraldehyde NADPH----glycerol ATP----glycerol 3-phosphate. All the enzymes of this pathway showed activity profiles commensurate with their participation in triacylglycerol synthesis which is maximal during the period 15-35 days after fertilization. Glycerokinase appears to be the rate-limiting enzyme in this pathway.     

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II)

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.     

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.     

Effect of magnesium ion on the structure of the 5S RNA from Escherichia coli. An imino proton magnetic resonance study of the helix I, IV, and V regions of the molecule

The imino proton nuclear magnetic resonance spectrum of Escherichia coli 5S ribonucleic acid (RNA) changes when the Mg2+ ion concentration drops below physiological levels. The transition between the physiological and low magnesium spectral forms of 5S RNA has a midpoint at approximately 0.3 mM Mg2+. Many of the most conspicuous changes observed in the downfield spectrum of 5S RNA as the magnesium concentration is reduced are due to adjustments in the structures of helices I and IV and the disappearance of resonances originating in helix V. The binding of ribosomal protein L25 to 5S RNA in the absence of magnesium stabilizes helix V structures.     

Bimodal oxygen uptake in a freshwater air-breathing fish,Notopterus chitala

Measurements of bimodal oxygen uptake have been made in a freshwater air-breathing fish,Notopterus chitala at 29.0±1(S.D.)°C. xhe mean oxygen uptake from continuously flowing water without any access to air, was found to be 3.58±0.37 (S.E.) ml O₂ · h^?1 and 56.84+4.29 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish weighing 66.92 + 11.27 (S.E.) g body weight. In still water with access to air, the mean oxygen uptake through the gills were recorded to be 2.49 ± 0.31 (S.E.) ml O₂ · h^?1 and 38.78 ± 1.92 (S.E.) ml O₂ · kg^?1 · h^?1 and through the accessory respiratory organs (swim-bladder) 6.04±0.87 (S.E.) ml O₂ · h^?1 and 92.32±2.91 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish averaging 66.92±11.27 (S.E.) g. Out of the total oxygen uptake (131.10 ml O₂ · kg^?1 · h^?1), about 70% was obtained through the aerial route and the remainder 30% through the gills.