Trematode myoglobins, functional molecules with a distal tyrosine.

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Solution of 1H NMR structure of the heme cavity in the oxygen-avid myoglobin from the trematode Paramphistomum epiclitum.

A two-dimensional 1H NMR study has been carried out on the heme cavity of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr, and 2 upfield ring current aliphatic side chains. Based solely on the conserved myoglobin folding topology that places the E helix-heme crossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix alignment were unambiguously established that place Tyr66 at position E7. Moreover, all eight aromatic and the two aliphatic side chains were shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr32(B10) and Tyr66(E7) rings indicate that both residues are oriented into the heme cavity, which is unprecedented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordinated oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autoxidation resistance of this myoglobin.

Structural and functional comparison of trematode haemoglobins.

Haemoglobins from three different species of trematodes (Gastrothylax crumenifer, Paramphistomum epiclitum and Isoparorchis hypselobagri) and their respective hosts were digested with papain. Peptides so produced were separated on 10-15% SDS-PAGGE. The probability of coincidence of the peptides was analysed statistically. Oxygen affinity curves of trematode haemoglobins were found to be a rectangular hyperbolic. The Hill coefficient values of all the trematode haemoglobins were found to be 1. The P50 values, at 25 degrees C in 0.2M phosphate buffer pH 7.4, ranged from 0.8 to 1.6 mmHg.

Purification and properties of the hemoglobins of the platyhelminth Isoparorchis hypselobagri (Trematoda: Isoparorchidae) and its host Wallagu attu (catfish).

1. The hemoglobins of the trematode Isoparorchis hypselobagri and of its host Wallagu attu (catfish) were isolated and purified. 2. SDS-polyacrylamide gel electrophoresis showed both to consist of single, 15-17 kDa chains, having different electrophoretic mobilities. 3. Isoelectric focusing showed the trematode hemoglobin to be homogeneous with a pI of 4.2 and the host hemoglobin to consist of several components. 4. Gel filtration of freshly prepared trematode hemoglobin revealed one peak corresponding to M(r) approximately 17 kDa; gel filtration of a preparation which had been stored for 2-3 months demonstrated the presence of two peaks, whose elution volumes corresponded to M(r) of ca 35 and 17 kDa, respectively. 5. Reversed-phase chromatography of carboxymethylated 35 and 17 kDa peaks on a C8 column, gave a single peak a and two peaks b and c, respectively. 6. Edman degradation of peaks a, b and c obtained provided identical sequences of 27 amino acid residues for peaks a and c and another sequence differing at 10 of the 27 positions, for peak b. Edman degradation of the freshly prepared Isoparorchis hemoglobin provided the first 15 amino acid residues found for peaks a and c. The host hemoglobin gave an N-terminal sequence completely different from the trematode sequences. 7. Since gel filtration of the 35 and 17 kDa peaks showed no sign of an interconversion equilibrium, it appears that the 35 kDa peak and peak a represent a disulfide-bonded dimer of a monomer globin chain which shares the 27 N-terminal residues with chain c, the major monomer globin component of the 17 kDa peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative spectral, electrophoretic and isoelectric properties of trematode haemoglobins.

A comparative study of the spectral, electrophoretic and isoelectric properties of the haemoglobins of three trematodes, Paramphistomum epiclitum, Gigantocotyle explanatum and Gastrothylax crumenifer was carried out. A high absorption in the beta band region indicates that trematode haemoglobins have high oxygen affinities. Electrophoretic mobilities of all trematode and their host haemoglobins were different. The isoelectric points of trematode haemoglobins were found to focus in the acidic range except that of G. crumenifer haemoglobin I, which focused at an alkaline pH.

Comparison of the hemoglobins of the platyhelminths Gastrothylax crumenifer and Paramphistomum epiclitum (Trematoda: Paramphistomatidae).

1. Gastrothylax crumenifer and Paramphistomum epiclitum parasitize the water buffalo Bubalus bubalis. 2. Gastrothylas hemoglobin consisted of two fractions of ca 30,000 and ca 18,000 by gel filtration. SDS-electrophoresis showed both to be single, ca 15,000 chains. 3. Paramphistomum hemoglobin was ca 16,000 by both gel filtration and SDS-electrophoresis. 4. Reversed-phase chromatography of carboxymethylated trematode and buffalo globins gave single peaks and two peaks, respectively. Although Paramphistomum hemoglobin provided and N-terminal sequence, Gastrothylax hemoglobin did not, suggesting blocked N-terminals. The buffalo sequences were found to be identical to the sequences of the alpha and beta chains of bovine hemoglobin. 5. Although Paramphistomum hemoglobin consists of only one chain, Gastrothylax hemoglobin consists either of one chain which aggregates to a dimer or of two different chains, only one of which aggregates to a dimer.

Lactate dehydrogenase in two digenetic trematodes and their host.

Polyacrylamide gel electrophoresis of the two digenetic trematodes, Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of the water buffalo, Bubalus bubalis revealed the presence of at least six and seven isoenzymes of lactate dehydrogenase (LDH), respectively in a partially purified enzyme preparation. The respective host tissues showed five isoenzymes of LDH, which are characteristic to the vertebrates. Both parachloromercuribenzoate and iodoacetate affected the LDH activity of the parasites and host tissues differently. Spectrophotometric analysis also showed different specific activity and susceptibility to the action of thiol inhibitors. The host LDH was quite stable at 57 degrees C for 30 min, but that of the parasites was less stable.

Characterization of sterols of three digenetic trematodes of buffalo.

Sterols of three digenetic trematodes were isolated and characterized by infrared and 1H nuclear magnetic resonance spectroscopies, and gas-liquid chromatography and gas chromatography-mass spectrometry. Sterols identified were cholesterol, cholestanol, 24-methylcholesterol, 24-methylcholestanol, 24-ethyl-22-dehydrocholesterol, 24-ethyl-22-dehydrocholestanol, 24-ethylcholesterol and 24-ethylcholestanol.

Histochemical and electrophoretic studies on phosphatases of some Indian trematodes.

The isoenzymes of acid and alkaline phosphatases and their histochemical localization were studied by polyacrylamide disc gel electrophoresis in four species of trematodes: Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of water buffalo (Bubalus bubalis) and Echinostoma malayanum and Fasciolopsis buski from the small intestine of the pig (Sus scrofa). Both acid and alkaline phosphatases were present in the tegument, gastrodermis, suckers, testes, ovary, eggs, vitellaria and uterus but alkaline phosphatase activity was demonstrated only in the parenchyma and excretory ducts. Polyacrylamide gel electrophoresis revealed two to four isoenzymes for both acid and alkaline phosphatase.

Health care resources and public policy in Pakistan.

PIP: Reviews the development of health care resources in Pakistan and their distribution. Inadequacies are found to be due to poor planning, capital shortages, and failure to consider sociocultural factors. Strengthening health care facilities is a primary responsibility of the government, requiring design and implementation of changes that will balance and integrate health care. Services must be appropriate to the circumstances, and based on the needs of the population.^ieng

Effects of some sugars, amino acids, glycerol and alpha-glycerophosphate on oxygen uptake of some digenetic trematodes.

Effect of various substrates on the oxygen uptake of some digenetic trematodes -- Srivastavaia indica, Gastrothylax crumenifer, Gigantocotyle explanatum from the buffalo, Bubalus bubalis and Isoparorchis hypselobagri from the fish Wallago attu was studied. In all the four species, the effect of various substrates except maltose was found to be stimulatory. The inhibitory or stimulatory effect of these substrates on oxygen uptake reflects the importance of these compounds in the energy metabolism. In spite of the fact that these trematodes were subjected to similar concentration of substrates, the percentage change in oxygen uptake is different in each of the trematodes, the differential utilization is probably related to the different ecophysiological conditions of the habitat of these trematodes. Such studies can be useful in devising better in vitro culture media for these trematodes.

Effect of anoxic incubation on the oxygen uptake of Isoparorchis hypselobagri (Digenea: Trematoda).

Anoxic incubation for varying periods of Isoparorchis hypselobagri from the swim bladder of the catfish Wallago attu, led to an accumulation of oxygen debt on the part of the parasite. With increasing length of anoxic incubation, there is a corresponding increase in post-anaerobic oxygen uptake, and the longer the period of incubation is, the greater is the time taken to return to a normal rate of oxygen uptake.

Quantitative studies on acetylcholinesterase in seven species of digenetic trematodes.

Quantitative estimation of absolute levels and in vitro release of acetylcholinesterase (AChE) in seven species of digenetic trematodes: Isoparorchis hypselobagri from the swim bladder of catfish, Wallago attu; Srivastavaia indica and Gastrothylax crumenifer from the rumen, and Gigantocotyle explanatum from the liver of the water buffalo, Bubalus bubalis; Fasciolopsis buski, Echinostoma malayanum from the small intestine and Gastrodiscoides hominis from the caecum of the pig, Sus scrofa revealed that the enzyme is present in remarkably high quantities in species which inhibit gastrointestinal tract compared with those that parasitize liver and swim bladder. The rate of in vitro release of AChE also varies with the species which supports the view that such differential secretion probably takes place in situ as well to counteract peristalsis and it is a biochemical adaptation on the part of these trematodes.

Alkali denaturation of oxyhaemoglobins of some digenetic trematodes and their hosts.

Oxyhaemoglobins of six digenetic trematodes,--Srivastavaia indica, Gastrothylax crumenifer, Gigantocotyle explanatum, Fasciolopsis buski, Gastrodiscoides hominis, Isoparorchis hypselobagri, and of their 3 vertebrate hosts, Bubalus bubalis, Sus scrofa, Wallago attu, were subjected to alkali denaturation at a pH of 12.4. All oxyhaemoglobins from trematodes and their hosts differ from each other in the rate and extent of alkali denaturation which may be explained due to variations in the amino acid sequences of a particular haem protein in addition to other factors.

Spectrophotometric analysis of haemoglobins of some digenetic trematodes and their hosts.

The haemoglobins of six different species of trematodes: Gastrothylax crumenifer, Srivastavaia indica, Gigantocotyle explanatum, Fasciolopsis buski, Gastrodiscoides hominis, Isoparorchis hypselobagri and their three different hosts: Bubalus bubalis, Sus scrofa, and Wallago attu were spectrophotometrically investigated, and were found to contain porphyrin IX as the common prosthetic group. Oxyhaemoglobin, carbonmonoxy-haemoglobin and reduced haemoglobin of all 6 species of trematodes and their 3 hosts under study gave similar absorption maxima. Distinct differences were, however, observed in the nature of the spectral curves of cyanmethaemoglobin which exhibit 2 absorption maxima in the beta and the alpha region in the case of all trematodes whereas in the case of similar host haemoglobin derivatives only one single broad peak in the 536-540 nm region was obtained. With respect to pyridine derivatives all the trematode haemoglobins show a sharp peak in the alpha region and a minor hump in the beta region except Gastrothylax crumenifer. All three host pyridine haemoglobin derivatives show only a single broad peak at 570 nm.

Qualitative analysis of substances excreted by Isoparorchis hypselobagri (Trematoda) during aerobic in vitro culture.

Isoparorchis hypselobagri from the swim bladder of the catfish, Wallago attu was aerobically cultured in vitro. The dialyzed incubate was microchemically analysed for either individual or group of compounds excreted by the parasite. Amino acids, amines, amides, ammonia, aldehyde, glycine, alpha-alanine, tyrosine, alpha-aminocarboxylic acids, haemoglobin, nitrogen, reducing materials, and lactic, pyruvic, succinic, propionic and acetic acids were found in the incubate, but acetaldehyde, formaldehyde, O-dioxo and oxomethylene, and formic acid were not detected.