Sheep Kidney Research Articles

β- d-Galactosidase isoenzymes in different sheep organs

1. 1. Two types of β- d-galactosidase activity are present in sheep organs. 2. 2. A β- d-galactosidase that binds to concanavalin A and has an acidic pH-optimum, characteristic of a lysosomal hydrolase, predominates in sheep brain, kidney and spleen. 3. 3. A different β- d-galactosidase that does not bind to concanavalin A is also present in these tissues and accounts for 95% of the β- d-galactosidase in sheep liver. 4. 4. β- d-Glucosidase, β- d-fucosidase, β- d-xylosidase and α- l-arabinosidase activities also do not bind to concanavalin A. 5. 5. All five activities that do not bind to concanavalin A show the same pH-dependence, thermal stability and inhibition by Cl − ions and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP), a specific β- d-glucosidase inhibitor. 6. 6. It is postulated that a broad specificity glycosidase which has greatest activity towards β- d-glucosides accounts for all these activities. 7. 7. As the true β- d-galactosidase is not inhibited by DMDP it will be possible to use this inhibitor in a differential assay for the two types of β- d-galactosidase in sheep with a deficiency of the lysosomal β- d-galactosidase.

Influence of dietary lipids on arrhythmias and infarction after coronary artery ligation in rats.

Coronary artery ligation (CAL) was used to produce an in vivo model of cardiac arrhythmias and myocardial infarction using anaesthetized male Hooded Wistar rats which had been fed for 6-7 or 18-20 months on either a standard reference diet alone or supplemented (12% w/w) with sunflower seed oil (linoleic acid rich) or sheep kidney fat (linoleic acid poor). The number of ventricular extra beats and duration of tachycardia or fibrillation in the 30-min postligation was increased in sheep kidney fat-fed rats. Infarct size 4 h postCAL was reduced in sunflower seed oil-fed rats. Arrhythmias, infarct size, and dietary-induced differences were increased with age. The diets employed produce changes in myocardial membrane phospholipids which could result in altered prostaglandin production. These results show that in the rat (as in man), age and dietary saturated fat are risk factors for sudden cardiac death and myocardial infarction and suggest that the rat is a useful model for the investigation of dietary interventions in heart disease.

Amino-acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA.

We have isolated and characterized a complementary DNA for the catalytic subunit of the sheep kidney sodium/potassium-dependent ATPase. The 1,016-amino-acid protein seems to have eight transmembrane domains. The apparent ouabain binding site is located at the extracellular junction of two transmembrane domains and is linked to the phosphorylation site by a 60-amino-acid conserved sequence that may be a major channel for energy transduction.

Use of synthetic oligonucleotide and recombinant DNA probes to study renin gene expression.

Using hybridization histochemistry renin gene expression has been localized in the juxtaglomerular apparatus (JGA) of the renal cortex in both mouse and sheep kidney. This technique also located renin gene expression in afferent arterioles and interlobular arteries distant from the glomerular tuft in lamb renal cortex. A short (30 mer) synthetic oligonucleotide probe, complementary to a region of the mouse submaxillary gland renin gene, specifically labelled mouse submaxillary gland and kidney. Hybridization histochemistry and Northern blot analysis using both the synthetic oligonucleotide (mouse) probe and a 700 base pair recombinant (sheep) probe showed differences in renin gene expression in the kidney in response to Na restriction in the mouse and Na depletion in the sheep.

Microneutralisation systems for use with different strains of peste des petits ruminants virus and rinderpest virus

Comparative studies were made to determine the most suitable microtitration system for assaying strains of peste des petits ruminants virus (PPRV) and rinderpest virus (RV). Infectivity titres did not differ significantly when assayed in either calf kidney, sheep kidney or Vero cells. However, cytopathic effects were much easier to detect in the latter making them the cell of choice. Addition of small amounts of virus to preformed cell monolayers in microplates with the subsequent addition of maintenance medium give higher infectivity titres than when cell suspension was added to virus, although the latter is more convenient for routine use. The titres of PPRV and neutralising antibodies assayed in tubes and microplates were not significantly different. Simultaneous screening of sera at a 1 in 20 dilution against both PPRV and RV gave a higher incidence of positives against homologous as opposed to heterologous virus.

266 GLUCONEOGENESIS BY THE FETAL SHEEP KIDNEY

We have consistently noted glucose release by the kidney of the unstressed fetal sheep, but it is not known whether this glucose is produced by gluconeogenesis or by glycogenolysis. To examine this question, we infused 14C-lactate intravenously into 5 healthy fetal sheep (mean gestational age 130 days) which had catheters chronically placed in the fetal descending aorta (FA), renal vein (RV), and inferior vena cava. We measured 14C-glucose and glucose concentrations in the RV and FA, total 14C-radioactivity and lactate concentration in the FA, and renal blood flow by the radioactive microsphere method. We calculated glucose flux across the kidney by the Fick method. Lactate specific activity was not measured; since total 14C-radioactivity may include 14C-metabolites of lactate, we could not calculate the actual quantity of glucose produced from lactate. We could, however, measure the minimum amount of glucose produced from lactate. We found net glucose release by the kidney (mean 3.0 mg/min/100 g kidney); at least 23% of this glucose was produced from lactate (0.7 mg/min/100g kidney).The unstressed fetal sheep kidney is therefore able to produce glucose by gluconeogenesis. This new glucose may be used by the kidney or may contribute to total fetal glucose supply. (Supported by HD 17618.)

Renin gene expression in vessels of the ovine renal cortex.

Using hybridization histochemistry, a technique which localizes specific mRNA populations in tissue sections with a 700 base pair recombinant DNA probe which codes for ovine renin, we have localized renin gene expression in the afferent arteriole of the juxtaglomerular apparatus (JGA) in the sheep renal cortex. Specific labelling representing renin gene expression was also found at a distance from the glomerular tuft in the walls of the afferent arteriole and also in cells in the medial layer of larger vessels of the renal cortex, specifically the interlobular arteries. These observations provide morphological evidence of renin gene expression at these sites and, combined with ultrastructural and immunocytochemical evidence suggest that renin is synthesized and stored in the afferent arteriole either within the JGA or at a distance from the glomerulus, and in the smooth muscle coat of the interlobular arteries in the sheep kidney.

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na + + K +)ATPase and sarcoplasmic reticulum Ca 2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n − 6 fatty acids, including linoleic (18:2(n − 6)) and arachidonic (20:4(n − 6)) while the sheep kidney fat dietary rats were higher in n − 3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n − 6/n − 3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.

Differential modulation of rat heart mitochondrial membrane-associated enzymes by dietary lipid

Diets supplemented with high levels of saturated fatty acids derived from sheep kidney (perirenal) fat or unsaturated fatty acids derived from sunflower seed oil were fed to rats and the effect on heart mitochondrial lipid composition and membrane-associated enzyme behaviour was determined. The dietary lipid treatments did not change the overall level of membrane lipid unsaturation but did alter the proportion of various unsaturated fatty acids. This led to a change in the ω6 ω3 unsaturated fatty acid ratio, which was highest in the sunflower seed oil fed rats. Arrhenius plots of the mitochondrial membrane associated enzymes succinate-cytochrome c reductase and oligomycin-sensitive adenosinetriphosphatase (ATPase) after dietary lipid treatment revealed different responses in their critical temperature. For succinate-cytochrome c reductase, the critical temperature was 29°C for rats fed the sheep kidney fat diet and 20°C for rats fed the sunflower seed oil diet. In contrast, no shift in the critical temperature for the mitochondrial ATPase was apparent as a result of the differing dietary lipid treatments. The results suggest that the discontinuity in the Arrhenius plot of succinate-cytochrome c reductase is induced by some change in the physical properties of the membrane lipids. In contrast, mitochondrial ATPase appears insensitive, in terms of its thermal behaviour, to changes occurring in the composition of the membrane lipids. However, the specific activity of the mitochondrial ATPase was affected by the dietary lipid treatment being highest for the rats fed the sheep kidney fat diet. No dietary lipid effect was observed for the specific activity of succinate-cytochrome c reductase. This differential response of the two mitochondrial membrane enzymes to dietary-induced changes in membrane lipid composition may affect mitochondrial oxidative phosphorylation.

A propos de l'utilisation des détergents SDS et saponine pour le démasquage et la purification de la (Na +K +)ATPase de rein de mouton

Incubation of membrane fragments from sheep kidney outer medulla in detergents such as SDS or saponin revealed increased specific (NA+) ATPase activity. Measurement of enzyme phosphorylation from [gamma 32P] ATP showed irreversible alteration of EP to (Na+) ATPase activity ratio when SDS is used as unmasking agent. These results were found with unmasked and purified enzyme.

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the ω6 to ω3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the ω6 to ω3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondria membrane-associated enzymes.

Initial-velocity kinetics of succinoyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney.

The initial-velocity kinetics of sheep kidney CoA-transferase are consistent with a Ping Pong mechanism. A KAcAc-CoA of 2.7 X 10(-5) M, KSucc-CoA of 1.6 X 10(-4) M, KSucc of 5.6 X 10(-3) M and KAcAc of 6.7 X 10(-5) M were determined by using a direct assay system that monitors the concentration of magnesium acetoacetyl-CoA enolate. However, product-inhibition kinetics of sheep kidney CoA-transferase are inconsistent with a Ping Pong mechanism. The possible involvement of separate binding sites for succinate and acetoacetate are discussed.

Differences in the fatty acid composition of atrial and ventricular phospholipids of rat heart following standard and lipid-supplemented diets

1. 1. The major saturated fatty acids of the phospholipids of rat heart atria and ventricles are similar and are not greatly altered by supplementing the diet with widely different types of lipid. 2. 2. There are important differences in the relative proportions of the major unsaturated fatty acids of the phospholipids of these anatomically and functionally distinct regions of the heart. 3. 3. The proportions of linoleic (C18:2, η-6) and docosahexaenoic (C22:6, η-3) acid are significantly higher in the ventricles than in the atria; the proportions of oleic (C18:1, η-9) arachidonic (C20:4, η-6) and docosatetraenoic acids (22:4, η-6) are higher in atria. 4. 4. The differences in unsaturated fatty acid profiles persist even after twelve months of feeding lipid supplements of sunflower seed oil (SSO) or sheep kidney (perirenal) fat (SKF). 5. 5. However, the ratios of arachidonic to docosahexaenoic acid in both tissues are changed by decreasing the intake of linoleic acid, which apparently favours the conversion of dietary linolenic (C18:3, η-3) to docosahexaenoic acid. The level of docosahexaenoic acid is greater in the ventricles than in the atria, and greatest when the animals were fed SKF diet. 6. 6. The physiological and pharmacological differences in ventricles and atria may arise from differences as fundamental as the phospholipid fatty acid composition of cardiac membranes.

Studies of the antigenic properties of sheep kidney Na +,K +-ATPase

Antibodies raised to the membrane Na +,K +-activated ATPase isolated from sheep kidney medulla were found to inhibit 60–70% of the ATPase hydrolytic activity, 30–40% of the K +-dependent p-nitrophenyl phosphatase activity, and to either inhibit or stimulate [ 3H]ouabain binding to the enzyme depending upon the ligands in the incubation media. A surface adsorption assay was used to detect the formation of antigen-antibody complexes with the immunizing antigen and with the lipids and the purified catalytic, glycoprotein, and proteolipid components of the enzyme. The lipid components are not antigenic; but the isolated, delipidated, and inactive catalytic and glycoprotein subunits are highly reactive with the holoenzyme-directed antibodies. The purified proteolipid, also, binds holoenzyme antibodies, however, at a reduced level and affinity (25%). Interestingly, the titers for the holoenzyme and the catalytic subunit are similar. In contrast, in “early” bleedings the antisera titer for the glycoprotein subunit is only 30% of that of the holoenzyme but in “late” bleedings it increases to about 60%. Competition binding studies provide the first direct evidence that the holoenzyme-directed antibodies are largely directed against native enzyme conformations which are retained by the isolated subunits and that 96 and 50–60% of the antibodies which bind to the isolated catalytic and glycoprotein subunits, respectively, also bind to the active enzyme. The proteolipid has now been demonstrated to be an antigenically active component of the purified enzyme but the extent to which complete recognition of its antigenic sites may depend upon conformational structures imposed by the ATPase complex is not known.

Characterization of ATP binding sites of sheep kidney medulla (Na + + K +)-ATPase using CrATP

The nucleotide substrate sites of sheep kidney medulla (Na + + K +)-ATPase are characterized using CrATP, a paramagnetic, substitution-inert substrate analogue probe. The paramagnetic effect of CrATP on 1 T 1 of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP in the presence of Na + and K + yielded characteristic enhancements for the binary enzyme-CrATP and ternary enzyme-Mg 2+-CrATP complexes of 3.3 and 3.6 and dissociation constants for CrATP of 5 and 12 μM, respectively. Substitution of Li + for K + in these titrations did not substantially alter the titration behavior. From the frequency dependence of 1 T 1 , the correlation time, τ c , for the dipolar water proton-CrATP interaction is 2.7 × 10 −10 sec, indicating that τ c is dominated by τ s , the electron spin relaxation time of Cr 3+. The paramagnetic effect of enzyme-bound Mn 2+ on 1 T 1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn 2+ complex with CrATP decreases the characteristic enhancement due to Mn 2+ from 6.6–8.0 to 1.5. The failure to observe free Mn 2+ epr signals in solutions of the ATPase, Mn 2+, and CrATP demonstrate that this decrease in ϵ Mn* is due to cross-relaxation between Mn 2+ and Cr 3+ bound simultaneously to the enzyme, and not to displacement of Mn 2+ from the enzyme by CrATP. The relaxation rate, 1⧸ T 1, of 7Li + is increased upon addition of CrATP to solutions of the ATPase, indicating that the sites for Li + and CrATP are close on the enzyme. A Cr 3+-Li + distance of 4.8 ± 0.5 Å is calculated from that data.

Growth potential of sheep and sea mammal cells transformed by SV40 early region DNA.

AbstractNormal fibroblasts of bearded seal, northern fur seal, sea lion, domestic sheep, and normal kidney cells of spotted dolphin and Pacific common dolphin have been transformed by fragments of SV40 DNA containing the entire early region. The transformed, T-antigen-positive cell strains grew to higher saturation densities and, except for the transformed dolphin kidney cell strains, were able, albeit poorly, to form colonies in soft agar medium. The transformed northern fur seal, spotted dolphin and Pacific common dolphin cell strains have remained diploid, the transformed sheep cells have become hypodiploid due to Robertsonian translocations, and the transformed bearded seal and sea lion cells had become hypotetraploid. Although all the transformed cells have acquired increased divisional potential and prolonged lifespan in cell culture, only the hypotetraploid cell strains of bearded seal and sea lion have evolved into continuous cell strains.

Isolation and partial characterization of an antigen associated with pregnancy in the ewe.

A precipitating antiserum raised in calves against sheep embryonic membranes was rendered specific for an antigen associated with pregnancy by absorption with sheep liver and kidney tissue. The Ouchterlony gel precipitin test was used to monitor the purification of ovine pregnancy-associated antigen (oPAA) from homogenized embryonic membranes. The antigen exhibited properties similar to known glycoproteins during conventional chromatographic and electrophoretic procedures. The purified antigen appeared to exist in high and low molecular weight forms (42,600 and 17,400 daltons, respectively) and the data suggested that these represented dimeric and monomeric forms of the antigen. The properties of this antigen are compared with those of other pregnancy-associated substances and the possible biological importance of the antigen is discussed.

Substrate-metal interactions at the active site of kidney (Na + + K +)-ATPase characterized by Mn(II) electron paramagnetic resonance

Electron paramagnetic resonance spectra at 35 GHz of Mn 2+ ion bound to highly purified membrane-bound (Na + + K +)-ATPase from sheep kidney medulla are much narrower than the corresponding spectra at 9 GHz. As a result, the sensitivity of the enzyme-Mn 2+ spectrum to added substrates is much greater at the higher frequency. ATP and AMP-PNP, which caused very little broadening at low frequency, effect dramatic decreases in intensity of the Mn 2+ EPR signal at 35 GHz. On the other hand, virtually no changes are observed upon addition of ADP and AMP, suggesting that the γ-phosphate of ATP plays a key role in the interaction between Mn 2+ and ATP on the enzyme. The data indicate that ATP and AMP-PNP, binding at low affinity substrate sites, induce a severe distortion of the Mn 2+ coordination geometry. The data also support the suggestion that the enzyme-bound Mn 2+ does not enter into a typical M 2+-ATP complex in this system.

Permeability of Plasma Membrane Vesicles to Ouabain and Mg2+ as a Factor Determining Rate of Binding of Ouabain to Na+ and K+ Dependent ATPase

Na+, K+-ATPase of the plasma membrane isolated from sheep kidney medulla exhibits functional asymmetry for the cardiac glycoside ouabain. In this vesicular membrane preparation the rate of binding of ouabain was slow (time constant greater than 60 min) when the vesicles were incubated in the presence of isotonic sucrose. Upon treatment of the preparation with hypoosmotic shock or phospholipase A the initial rate of ouabain binding was enhanced at least 3 fold. In equilibrium a concentration of the ouabain-enzyme-complex was obtained which was about twofold that of the untreated vesicles. This result suggests two types of ouabain binding sites with an approximate stoichiometry of 1 to 1. The stoichiometry seems to be maintained at high concentrations of ouabain where binding curves show a biphasic time course. Additional information about heterogeneity of binding sites comes through experiments in which the vesicles were treated with Mg2+ prior to the addition of ouabain. A minor fraction of the binding sites were occupied by ouabain only after longtime incubation with Mg2+.

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.