Potential and current distribution in an ideal Hall bar.
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Journal Article↗
Biomedical subjects
J Hajdu
Publications and source records attributed to J Hajdu.
Structure determination of turkey egg-white lysozyme using Laue diffraction data.
The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C alpha positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in catalysis (Asp52 and Glu35) are accessible to solvent and not blocked by crystal contacts.
Animals↗
Activity of crystalline turkey egg white lysozyme.
Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.
Animals↗
Phospholipids containing nitrogen- and sulfur-linked chains: kinetics of cholesterol exchange between vesicles.
We have examined the kinetics of [14C]cholesterol exchange between unilamellar vesicles formed from the following synthetic glycerophosphatidylcholines: (a) those having acyl (OC(O)R), acylamino (NHC(O)R), carbamoyl (NHC(O)OR), and acylthio (SC(O)R) chains at the sn-2 position, and (b) those having alkyl (OR) and thioalkyl (SR) chains at the sn-1 position. Replacement of the glycerol oxygen atom at the sn-2 position of PC with a NH group did not affect the rate of cholesterol exchange to a significant extent, suggesting that the amide group of sphingomyelin is not primarily responsible for the very slow rate of exchange of cholesterol observed from sphingomyelin vesicles. Replacement of the glycerol oxygen at the sn-2 position of phosphatidylcholine with a sulfur atom caused the rate of spontaneous cholesterol exchange to increase by a factor of 1.6. Substitution of an O-alkyl chain for the acyl chain at the sn-1 position of 2-acylthiophosphatidylcholine or substitution of a thioalkyl chain for the O-alkyl sn-1 chain of 1-alkyl-2-acylaminodeoxyphosphatidylcholine also did not result in a marked difference in cholesterol exchange rate. The data suggest that interactions other than intermolecular hydrogen bonding are involved in determining the rates of intermembrane cholesterol exchange. Significantly, these kinetic studies also lend support to the continued use in model membranes of synthetic sulfur- and nitrogen-substituted phosphatidylcholines, which have been employed to study properties of lipolytic enzymes, since synthetic acylamino- and acylthio-phospholipids form vesicles that give cholesterol exchange rates that closely resemble those found in vesicles prepared with diester-phosphatidylcholines.
1,2-Dipalmitoylphosphatidylcholine↗
Towards time-resolved diffraction studies with glycogen phosphorylase.
Laue diffraction with high intensity, broad-spectrum synchrotron radiation sources allows three-dimensional data sets on protein crystals to be recorded in seconds or milliseconds and opens the way for time-resolved studies on dynamic events in crystals. This chapter briefly reviews the field and describes progress towards time-resolved studies with glycogen phosphorylase. Methods for the synchronization of the start of reaction with the start of data collection have been developed for the phosphorolytic reaction of glycogen phosphorylase. The compound 3,5-dinitrophenylphosphate is photolabile, yielding Pi and the by-product, 3,5-dinitrophenol, which is non-reactive with the enzyme. Spectroscopic studies show that the compound has good quantum yield and that photolysis is rapid (greater than 1000 s-1). Release of the dinitrophenylate anion, following a pulse of light from a xenon flash lamp, has been monitored with a diode array spectrophotometer specially adapted for measurements on crystals. In a laboratory X-ray experiment with crystals of glycogen phosphorylase b, release of Pi and formation of the enzyme-product complex have been demonstrated. The way is now open for Laue diffraction studies on the catalytic reaction in the crystal.
Crystallography↗
Propranolol:methacrylic acid copolymer binding interaction.
The polyanionic form of methacrylic acid:methacrylic acid methyl ester copolymer reacts readily with propranolol.HCl to give a sparingly soluble complex at saturation equilibrium. Propranolol: methacrylic acid copolymer complex is characterized by differential thermal analysis, and IR and UV spectroscopic methods. Propranolol content is found to be 68% in the complex. The value of the Hill coefficient (1.5) indicates that there is a high degree of positive cooperative interaction between propranolol and the polymer.
Allosteric Regulation↗
Comparison of selective cytotoxicity of alkyl lysophospholipids.
Alkyl lysophospholipids have been shown to be cytooxic to a number of neoplastic tissues. One, ET-18-OCH3, has been used to selectively purge leukemic cells from mixtures with normal marrow progenitor cells, in vitro and in vivo. We have measured the 50% inhibitory (IC50) effect of a series of ether lipids (EL) on leukemic cells (HL60, K562, Daudi, KG-1, KG-1a) and normal marrow progenitor cells. Cells were incubated with varying concentrations of EL for 4 hr and assayed for viability, [3H]thymidine incorporation and clonogenicity in semi-solid media. The effect on protein kinase C (PKC) activity was assayed for each compound. Compounds tested included three glycerophosphocholine analogs--ET-18-OCH3, ET-16-NHCOCH3, and BM 41.440. In addition, a lipoidal amine, CP 46665, an ethyleneglycolphospholipid, AEPL, and four single chain alkylphosphocholine analogs, HePC2, HePC3, HePC4 and HePC6 were also tested. During the period of incubation, the cells remained viable (greater than 70%) as judged by trypan blue dye exclusion. The glycerophosphocholines were the most active and showed the highest therapeutic index. The lipoidal amine was active, but toxic to normal marrow progenitor cells. The ethyleneglycolphospholipid was active against HL60, but not against the other cell lines. The single chain alkylphosphocholine analogs were less active. All of the compounds inhibited PKC activity; however, the glycerophosphocholines were the most inhibitory.
Antineoplastic Agents↗
Stereospecific synthesis of antitumor active thioether PAF analogs.
A novel stereospecific synthesis of antitumor active thioether analogs of platelet-activating factor (PAF) is reported. The synthesis is based upon: i) the use of D-serine to provide the chiral center for the construction of the optically active phospholipid molecule; ii) development of the sn-1-thioalkyl function via thioacetate displacement of methanesulfonate-activated primary hydroxyl group followed by alkylation of the sn-1-thiolate function; and iii) introduction of the phosphocholine moiety through the 2-chloro-2-oxo-1,3,2-dioxaphospholane/trimethylamine sequence. The entire scheme relies on the use of a single protecting group. The synthetic thioether phospholipid 1-S-hexadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine has been shown to be a potent antitumor active phospholipid, exhibiting tumor cytotoxicity against a lymphoblastoid lymphoma (Li-A) cell line and a malignant histiocytic (DHL-4) cell line of human origin at the same level of potency as ET-18-OMe and 1-O-octadecyl-2-N-acetamidodeoxy-sn-glycero-3-phosphocholine. The synthetic method described has a great deal of flexibility, providing a convenient general route to a wide range of thioether PAF analogs.
Antineoplastic Agents↗
Calcium binding sites in tomato bushy stunt virus visualized by Laue crystallography.
We have collected Laue diffraction data from crystals of tomato bushy stunt virus using the full white X-ray spectrum from the wiggler magnet of the Synchrotron Radiation Source at Daresbury, U.K. A single 24 second exposure of a crystal soaked in EDTA yielded a data set that was 90% complete between 6 and 3.5 A resolution. A large proportion of the data could be measured using an overlap deconvolution routine to separate spatially overlapping reflections in the dense Laue photograph. Reflections with I greater than 2 sigma I (40% of the data set) were subjected to wavelength normalization. A difference Fourier map between these reflections and a monochromatic native set showed, after icosahedral averaging, the three pairs of Ca2+ binding sites related by quasi-symmetry and the movement of a liganding loop in the protein at the A/C subunit interface. The extent and quality of the data obtained from a single Laue photograph of this virus were thus sufficient to detect clearly such small structural alterations. In a second experiment, a Laue photograph was taken from a crystal that was soaked first in EDTA and then in GdCl3. A difference Fourier map between this Laue data set and the Laue data set from the EDTA-soaked crystal showed clearly the Gd3+ sites in the capsid, demonstrating that the Laue technique is a reliable and efficient means for data collection with virus crystals.
Binding Sites↗
Inhibition of protein kinase C, (sodium plus potassium)-activated adenosine triphosphatase, and sodium pump by synthetic phospholipid analogues.
The effects and modes of action of certain antineoplastic phospholipid analogues (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine, BM 41.440, JH-1, CV-3988, and HePC) on (sodium plus potassium)-activated adenosine triphosphatase (Na,K-ATPase) and sodium pump activities were investigated. Inhibition of Na,K-ATPase in purified rat brain synaptosomal membranes by these lipids, in contrast to ouabain, was subject to membrane surface dilution and unaffected by whether the reaction was started with KCl, NaCl, or ATP. Kinetic analysis indicated that the analogues, again dissimilar to ouabain, were likely to interact directly or indirectly with sodium-binding sites of Na,K-ATPase located at the intracellular surface of the plasma membrane, a conclusion also supported by studies using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not the lipids) increased the affinity constant of Na,K-ATPase for K+, whereas the lipids (but not ouabain) increased that for Na+. The lipids also inhibited 86Rb uptake by intact human leukemia HL60 cells at potencies quite comparable to those seen for inhibition of purified protein kinase C or Na,K-ATPase. It is suggested that Na,K-ATPase (sodium pump) might represent a hitherto unrecognized site of action for the lipid analogues, and that the antineoplastic effects of the agents might be due to, in part, inhibition of both protein kinase C and Na,K-ATPase and perhaps other membrane-associated enzymes.
Biological Transport, Active↗
Progress with Laue diffraction studies on protein and virus crystals.
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Lasers↗
The interaction of phospholipase A2 with phospholipid analogues and inhibitors.
A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)
Binding Sites↗
Phospholipids showing complex bilayer phase transitions. II. Four sulfur-containing phosphatidylcholines.
The main phase transitions of aqueous dispersions of four synthetic phosphatidylcholines (PCs) containing sulfur atoms in thioester and thioether linkages have been studied by high sensitivity differential scanning calorimetry (DSC). The transition enthalpies ranged from 7.4 to 10.3 kcal mol-1, with values for tm, the temperature of maximal excess heat capacity, in the range 38.0 to 40.8 degrees C. The corresponding values for dipalmitoyl PC (DPPC) are 8.5 kcal mol-1 and 41.7 degrees C. Curve fitting required the sum of from one to four two-state components to give an accurate representation of the observed DSC curves. Comparison of the results given here with those reported by B.Z. Chowdhry, G. Lipka, J. Hajdu and J.M. Sturtevant, (1984) Biochemistry 23, 2044-2049, for PCs containing amide, ether or carbamoyl linkages in place of the usual ester bonds shows that small changes in organic structure can result in large changes in thermotropic behavior. The complexity in the cases showing more than a single two-state component is presumably due to a series of sequential cooperative transitions the character of which is at present unknown.
Calorimetry, Differential Scanning↗
Laue crystallography: application to virus crystals.
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Crystallography↗
Channels at the catalytic site of glycogen phosphorylase b: binding and kinetic studies with the beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane.
Regions of low packing density in the vicinity of the catalytic site of glycogen phosphorylase b are described with the aid of a computer program that generates a contour map in which the contour level is inversely proportional to the packing density in the protein. It is shown that, although there is no direct route from the catalytic site to the surface, there are two possible channels that could allow access for substrates following conformational changes in the enzyme. The first channel, channel 1, leads from the catalytic site to the surface close to the nucleoside inhibitor site and requires movements of residues 280-285 and Arg 569 in order to obtain access. Previous crystallographic experiments have shown that in the presence of substrates or R-state inhibitors these parts of the polypeptide chain undergo large conformational changes. The properties of the second channel (channel 2), which is the more extensive channel, have been investigated with the potent beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane (PUG). Crystallographic binding studies at 2.4-A resolution show that the compound binds neatly at the catalytic site of phosphorylase b. The glucopyranosylidene ring, in the half-chair conformation, occupies a similar but not identical position (shift about 0.6 A) to that occupied by other glucosyl compounds bound at the catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
[Online computerized data processing in a perinatal intensive care unit].
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Electronic Data Processing↗
1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine, a highly specific chromogenic substrate of phospholipase A2.
1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine (2-thioPC), a structurally modified phospholipid analog is specifically hydrolyzed by phospholipase A2 to liberate 2-thiolysophosphatidylcholine and palmitic acid. The sulfhydryl group of the product is readily trapped by 5,5'-dithiobis (2-nitrobenzoic acid) allowing continuous spectrophotometric monitoring of the enzymatic reaction. The rates of hydrolysis by bee-venom phospholipase A2 have been determined in a series of Triton X-100 containing mixed micelles. At 1 mM 2-thioPC increasing the concentration of Triton X-100 from 4 to 16 mM changes the specific activity of bee-venom phospholipase A2 from 96.9 to 17.9 mumol/min/mg, about one order of magnitude lower than dipalmitoyl phosphatidylcholine hydrolysis in micelles of similar composition. The chromogenic substrate is the first phospholipid analog exhibiting absolute specificity for phospholipase A2 and should be applicable to spectrophotometric detection and kinetic characterization of both water soluble and membrane-bound forms.
Animals↗
Preformulation study of pelrinone hydrochloride.
Pelrinone HCl is essentially nonhygroscopic. The pH-solubility profile exhibits a U-shaped curve, while the octanol-water partition coefficient-pH profile shows a bell-shaped curve. Two ionizable functions, with a pKa1 value of 4.71 and a pKa2 value of 8.94, produce the cationic and anionic forms, respectively. A weak ionic strength effect on solubility of the compound is observed: at pH 3.9 (0.1 M acetate buffer), the solubility increases with increasing ionic strength, while at pH 7.5 (Tris HCl buffer), the solubility decreases with increasing ionic strength. No gross incompatibility of the compound is seen with the 13 excipients selected, except povidone. The solubility phase diagram, X-ray diffraction pattern, and IR spectroscopy demonstrate the presence of polymorphs. The compound in solution is stable at various pH conditions under 500-foot-candle (ft-c) light at room temperature and at 80 degrees C for 64 d. In the solid state, no decomposition is observed at 80 degrees C and on exposure to 500-ft-c light for at least 112 d.
Cardiotonic Agents↗