Demonstration of acid phosphatase in Eimeria spp.: partial characterization of the enzyme in E. vermiformis

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporulated oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chromatography.     

Coccidiosis of the wild rabbit (Oryctolagus cuniculus) in France

In 1998-1999 a survey of coccidiosis in wild rabbits was carried out in six different localities in France. About five individuals were caught monthly in each locality and a total of 254 wild rabbits was examined. Ten species of Coccidio were identified: Eimeria perforans, E. flavescens, E. piriformis, E. exigua, E. media, E. magna, E. coecicola, E. stiedai, E. roobroucki, E. intestinalis. Intensity of infection in young individuals was higher than in adults. Intensity was highest in winter but, as there are no young rabbits in winter, in young individuals it was higher in spring and autumn than in summer. Intensities were higher in the northern rather than in southern localities. Ranking of prevalence was remarkably stable, in contrast to the variability of the parasitic load. The equilibrium between congeneric species of rabbit coccidia (stable prevalence rank, variable parasitic load) is thought to be probably the consequence of the opportunistic feeding habits of rabbits.     

Further studies on the properties of glucose 6-phosphate dehydrogenase from the coccidium Eimeria stiedai (Protozoa).

1. Glucose 6-phosphate dehydrogenase from Eimeria stiedai does not reduce NAD or any of its analogs tested. It does reduce NADP and its thionicotinamide and 3-acetylpyridine analogs. 2. It will accept D-glucose as substrate, but not 2-deoxy-D-glucose, glucose 1-phosphate, or 2-deoxy-D-glucose 6-phosphate. 3. Its response to a number of compounds that activate or inhibit the enzyme from other organisms has been determined. 4. The molecular weight is ca. 240,000 by gel chromatography, and only one isoenzyme could be detected by disc electrophoresis. 5. The enzyme resists conditions that commonly cause dissociation to lighter weight active forms.     

The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema.

Large chondroitinsulphate-containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP-9 and proMMP-2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP-2 and MMP-9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.     

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.     

Factors influencing the fecal egg and oocyst counts of parasites of wild European rabbits Oryctolagus cuniculus (L.) in Southern Western Australia

Abundance of intestinal parasites was monitored by fecal egg and oocyst counts for samples of wild rabbits Oryctolagus cuniculus with different levels of imposed female sterility from 12 populations in southwestern Australia. Differences in egg counts of Trichostrongylus retortaeformis between seasons and age groups were dependent on the sex of the host. Pregnancy may have been responsible for these differences because egg counts were consistently higher in intact females than in females surgically sterilized by tubal ligation. Egg counts for Passalurus ambiguus were influenced by season and host age but there were no differences between sexes or between intact and sterilized female rabbits. No differences were detected in the oocyst counts of the 8 species of Eimeria between male and female rabbits or between intact and sterilized females. Seasonal differences were detected in oocyst counts of Eimeria flavescens and Eimeria stiedai. The overwhelming determinant of coccidian oocyst counts was host age, with 6 species being much more abundant in rabbits up to 4 mo of age. There was a suggestion that egg counts of T. retortaeformis and oocyst counts of several species of Eimeria were reduced in populations where rabbit numbers had been depressed for at least 2 yr, but there was no evidence that short-term variations in rabbit numbers had a measurable effect on parasite abundance.     

Prevalence and pathological study on rabbit hepatic coccidiosis in Taiwan.

Five breeds of rabbits, which included the New Zealand, Californian, Spot, Rex and Angora rabbit, were found from a survey of 1,152 rabbits in Taiwan. The prevalence of coccidia in young rabbits (weaning-2 months old) was 95% to 100%. Adult female rabbits usually acted as carriers within the farm and transmitted the parasite to young rabbits, which caused severe infection with clinical signs and even death. Parasitism of hepatic coccidia (Eimeria stiedai) in the rabbit led to severe mortality. Numerous and scattered white nodules about 0.1 to 0.5 cm in diameter were seen on the liver surface and dark greenish mucoid exudate was found in intestinal lumen. Histopathologic lesions included hyperplasia of the bile duct epithelium with different developmental stages of coccidia within. Oocysts could be seen in the lumen, and granuloma tissues encircle the bile duct with infiltration of inflammatory cells. The other organs were not infected.     

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.     

Crystal structure of sarcoplasmic reticulum Ca2+-ATPase (SERCA) from bovine muscle

The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9? resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Different sensitivity of the sarcoplasmic reticulum Ca(2+)-ATPase enzyme to fluorescein-isothiocyanate in rabbit and carp muscles.

1. Carp and rabbit sarcoplasmatic reticulum Ca(2+)-ATPase enzymes were compared with respect to their sensitivity to FITC labelling. 2. The carp enzyme showed much lower sensitivity to FITC in the Ca(2+)-Mg2+ activated ATPase activity. Fifty percent inhibition was observed at 20 microM labelling FITC concentration; in rabbit enzyme this inhibition was already achieved at 2 microM FITC. 3. The tryptic cleavage products of the carp enzyme identified with immunoblot analysis as well as with FITC fluorescence, suggest multiple cleavage, yielding different fragments from the ones well known in rabbit and in rat enzyme. 4. The present results indicates major structural differences with respect to the FITC binding, and tryptic cleavage between the SR Ca(2+)-ATPase enzymes from carp and rabbit, despite the cross-reactivity with polyclonal antibodies.     

Regional mapping of the creatine kinase b (CKBB) gene in rabbit (Oryctolagus cuniculus) and man using a rat cDNA probe

Using a rat creatine kinase (brain form) cDNA clone for in situ hybridization, we have localized the gene in both the human and the rabbit complement. An analysis of the data shows that the locus in the human is at 14q32, confirming previous assignments based on somatic hybridization studies and Southern blot analysis. In the rabbit, significant accumulation on 20q13----qter with the predominant labeling at the end of the chromosome provides evidence for the localization of the gene at this site. The heterologous hybridizations of a rat probe to both human and rabbit metaphases underscore the highly conserved nature of the sequences for this enzyme.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Eimeria stiedai in rabbits: the presence of an oocyst residuum

Observations on 5 different isolates of Eimeria stiedai from pure infections in laboratory-reared rabbits have confirmed the presence of an oocyst residuum. This structure was seen in 89--100% of the oocytes in cultures prepared both from the liver and from the faeces at different times during the patent period. The residuum consisted of 1--7 or more granules situated centrally in the oocyst and partially concealed by 4 sporocysts. The differential diagnosis of E. stiedai and E. coecicola is discussed.     

Biochemical differentiation between the Wild rabbit (Oryctolagus cuniculus L.), the Domestic rabbit and the Brown hare (Lepus europaeus Pallas)

Twenty five specimens of the brown hare and fourteen specimens of the wild rabbit were examined for genetic differentiation and polymorphism in 28 enzyme systems by means of horizontal starch gel electrophoresis and isoelectric focusing in polyacrylamide gels. Indices of genetic variation show values similar to those averaged over numerous mammalian species. A D-value of 0, 0288 between wild and domestic rabbits suggests a high amount of gene frequency changes at enzyme loci during domestication. Evolutionary time of divergence between the rabbit and the hare (about 2430000 years) calculated from electrophoretic data is in good agreement with palaeontological findings.     

Activity and function of rabbit muscle-specific creatine kinase at low temperature by mutation at gly268 to asn268

Carp muscle-specific creatine kinase M1 isoenzyme (M1-CK) seems to have evolved to adapt to synchronized changes in body temperature and intracellular pH. When gly(268) in rabbit muscle-specific creatine kinase was substituted with asn(268) as found in carp M1-CK, the rabbit muscle-specific CK G286N mutant specific activity at pH 8.0 and 10°C was more than 2-fold higher than that in the wild-type rabbit enzyme. Kinetic studies showed that K(m) values of the rabbit CK G268N mutant were similar to those of the wild-type rabbit enzyme, yet circular dichroism spectra showed that the overall secondary structures of the mutant enzyme, at pH 8.0 and 5°C, were almost identical to the carp M1-CK enzyme. The X-ray diffraction pattern of the mutant enzyme crystal revealed that amino acid residues involved in substrate binding are closer to one another than in the rabbit enzyme, and the cysteine283 active site of the mutant enzyme points away from the ADP binding site. At pH 7.4-8.0 and 35-10°C, with a smaller substrate, dADP, specific activities of the mutant enzyme were consistently higher than the wild-type rabbit enzyme and more similar to the carp M1-CK enzyme. Thus, the smaller active site of the RM-CK G268N mutant may be one of the reasons for its improved activity at low temperature.     

Interaction of eukaryotic threonyl-tRNA synthetase with high-Mr RNAs and tRNA(Thr)

Threonyl-tRNA synthetase of rabbit reticulocytes was purified to homogeneity. We have found that this enzyme can interact not only with cognate tRNA(Thr), but also with high-Mr RNAs. tRNA(Thr) removes rRNA from the complexes with threonyl-tRNA synthetase. On the other hand, rRNA is unable to dissociate tRNA(Thr) from the complexes with the enzyme. Despite its dimeric organization, threonyl-tRNA synthetase is unable to form stable ternary complexes with tRNA(Thr) and rRNA. In the extract of rabbit reticulocytes about one-third of the threonyl-tRNA synthetase molecules are in association with cognate tRNA(Thr) and thus are unable to interact with high-Mr RNAs.     

Isolation and immunological characterization of three highly purified serine proteinases from Antarctic krill (Euphausia superba)

Summary Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.     

Species distribution and properties of hepatic phenylalanine (histidine):pyruvate aminotransferase.

Hepatic phenylalanine(histidine):pyruvate aminotransferase activity is much higher in the mouse and rat than in other animal species (human, guinea-pig, rabbit, pig, dog and chicken). The activity is elevated in the mouse and rat by the injection of glucagon but not in other species (guinea-pig, rabbit and chicken). The enzyme was purified from the mitochondrial fraction of mouse liver to homogeneity as judged by polyacrylamide disc gel electrophoresis in the presence of dodecylsulphate. With histidine as amino donor, the enzyme was active with pyruvate, oxaloacetate and hydroxypyruvate as amino acceptors but not with 2-oxoglutarate. Effective amino donors were histidine, phenylalanine and tyrosine with pyruvate, and methionine, serine and glutamine with phenylpyruvate. The apparent Km for histidine was about 6.9 mM with pyruvate and that for pyruvate was 21 mM with histidine. The enzyme is probably composed of two identical subunits with a molecular weight of approximately 40000. The pH optimum was near 9.0. Isoelectric focusing of the purified enzyme resulted in the detection of four forms with pI 6.0, 6.2, 6.5 and 6.7, respectively, all of which were responsive to glucagon. These four forms were nearly identical with the purified enzyme before the focusing with respect to physical and enzymic properties. A possible mechanism of this multiplicity is discussed.     

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2)

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.