Evidence for selenocysteine in ovine tissue organelles

Subcellular fractions were prepared by differential centrifugation from heart and liver of a lamb labeled with 75Se-selenite. Crude fractions of nuclei, mitochondria, and microsomes from both tissues were solubilized with sodium dodecyl sulfate and chromatographed on columns of sephacryl S-200. A low molecular weight (MW) 75Se labeled cardiac cytosol protein (approximately 10,000 daltons) was partially purified by gel filtration chromatography. The major 75Se peaks from the sephacryl columns and the low MW cardiac protein were hydrolyzed in HCl under an inert atmosphere. When chromatographed on an amino acid analysis column, 75Se from each hydrolysate chromatographed in the identical position of 2,7-diamino-4-thia-5-selenaoctanedioic acid, the mixed oxidized dimer of cysteine and selenocysteine. The low MW cardiac protein was reacted with chloroacetate after reduction with borohydride. 75Se from a hydrolysate of this derivatized protein eluted in the same position as Se-carboxymethylselenocysteine on the amino acid analysis column. Thus, selenocysteine appears to be the predominant form of selenium in ovine heart and liver.     

Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in commercial-line turkeys

The objective of this study was to determine the concentration of total selenium (Se) and proportions of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in the tissues of female turkeys offered diets containing graded additions of selenized-enriched yeast (SY), or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of breast and thigh muscle were assessed at 0 and 10 days post mortem. A total of 216 female turkey poults were enrolled in the study. A total of 24 birds were euthanized at the start of the study and samples of blood, breast, thigh, heart, liver, kidney and gizzard were collected for determination of total Se. Remaining birds were blocked by live weight and randomly allocated to one of four dietary treatments (n = 48 birds/treatment) that differed either in Se source (SY v. SS) or dose (Con [0.2 mg/kg total Se], SY-L and SS-L [0.3 mg/kg total Se as SY and SS, respectively] and SY-H [0.45 mg total Se/kg]). Following 42 and 84 days of treatment 24 birds per treatment were euthanized and samples of blood, breast, thigh, heart, liver, kidney and gizzard were retained for determination of total Se and the proportion of total Se comprised as SeMet or SeCys. Whole blood GSH-Px activity was determined at each time point. Tissue GSH-Px activity and thiobarbituric acid reactive substances were determined in breast and thigh tissue at the end of the study. There were responses (P < 0.001) in all tissues to the graded addition of dietary Se, although rates of accumulation were highest in birds offered SY. There were notable differences between tissue types and treatments in the distribution of SeMet and SeCys, and the activity of tissue and erythrocyte GSH-Px (P < 0.05). SeCys was the predominant form of Se in visceral tissue and SeMet the predominant form in breast tissue. SeCys contents were greater in thigh when compared with breast tissue. Muscle tissue GSH-Px activities mirrored SeCys contents. Despite treatment differences in tissue GSH-Px activity, there were no effects of treatment on any meat quality parameter.     

Selective precipitation of haptoglobin and alpha2-macroglobulin from human serum using Alocasia macrorhiza tuber protein

Treatment of human serum with ammonium sulfate fraction (0-50%) of Alocasia macrorhiza tuber extract resulted in precipitation at neutral pH. The precipitate was dissolved at pH 10.5 and chromatographed on Sephadex G-100 column. Two protein peaks were resolved. While the first peak represented alpha2-macroglobulin and haptoglobin, the second peak accounted for specific Alocasia protein. Incidentally the Alocasia protein was shown to be responsible for selective and specific precipitation of alpha2-macroglobulin and haptoglobin from serum. Thus the plant protein in its pure form or in crude stage could be used for the rapid isolation of two of the prominent alpha2-globulins.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Separation of two molecular forms of rat adipose-tissue lipoprotein-lipase.

Lipoprotein-lipase extracted from rat adipose tissue acetone-ether powders, was separated by gel filtration on Biogel A 1.5 M into two active fractions: lipoprotein-lipase “a” and lipoprotein-lipase “b” as named by Garfinkel et al. (1). Then each of these two fractions was again chromatographed on heparin-Sepharose column according to our own method (2). The lipoprotein-lipase “b” was eluted in the first fraction like the microsomal lipoprotein-lipase; lipoprotein-lipase “a” was eluted in the second one like plasma membranes lipoprotein-lipase (3). We compared the properties of the two lipoprotein-lipases obtained by those two different chromatographic methods.     

Effect of dietary selenium restriction on selected parameters of selenium status in men with high life-long intake

The influence of selenium (Se) restriction on disposition in plasma and urine fractions of infused (74)Se (selenite) was studied when adult males (Enshi City, Hubei Province, PRC) whose habitual daily Se intake is approximately 480 microg per day were transferred to Lichuan County, where the daily intake is approximately 30 microg. The subjects received an infusion (106 microg Se) on the day before consuming foods low in Se and a second infusion (113 microg Se) 63 days later. Blood and 24-hour urine samples were collected each day for 7 days after the first infusion and on days 22, 43, and 62 following the first infusion. Urine and blood were also collected daily for the next 7 days after the second infusion. Plasma total Se concentration increased for 7 days after each of the two infusions and urine Se decreased exponentially following both the first and second infusions. The excretion of trimethylselenonium followed the same pattern as the total urinary Se. Surprisingly, there was not a significant difference in selenite retention between the two infusion periods, and the data indicated that, regardless of the chemical form of Se present in various organs, its catabolism leading to excretion in urine followed the same pathway as that of selenite. Labeled Se was incorporated predominantly in the plasma selenoprotein P fraction and the half-life of Se in this fraction was determined to be 1.9 to 2.9 days. Thus, a longer depletion period is required in these subjects to obtain more significant changes.     

Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro.

Following the simultaneous subcutaneous administration of CdCl2 and Na2SeO3 to rats, evidence of a Cd-Se complex was detected in plasma by gel filtration chromatography. A similar complex was found in plasma after incubation of selenite, Cd, rat erythrocytes, and plasma in vitro, and after incubation of H2Se, Cd, and plasma in vitro. No interaction of selenite, selanete, or selenodiglutathione with Cd and plasma in the absence of erythrocytes in vitro was noted. Characterization by gel filtration, ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation showed that these Cd-Se complexes are similar. The results support the hypothesis that H2Se or a similarly reduced selenide is the product of selenite metabolism by rat erythrocytes. Hydrogen selenide also altered the distribution of inorganic mercury in rat plasma in vitro in such a way that the apparent molecular weights of the Se-Hg and Cd-Se complexes associated with protein were similar. Hydrogen selenide had no effect upon the distribution of methylmercury in plasma. The stability of the Cd-Se complex in plasma depended upon the integrity of the native protein components, as shown by incubation with Proteinase K. The properties of the complex suggested that it existed in a single form associated with different plasma components under various conditions.     

Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma.

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.     

Effects of dietary selenium supplementation on tissue selenium distribution and glutathione peroxidase activity in Chinese Ring Necked Pheasants

The objective of this study was to determine the concentration of total selenium (Se) and the proportions of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in the postmortem tissues of female pheasants (Phasianus Colchicus Torquator) offered diets that contained graded additions of selenised-enriched yeast (SY) or a single comparative dose of sodium selenite (SS). Thiobarbituric acid reactive substances (TBARS) and tissue glutathione peroxidase (GSH-Px) activity of breast (Pectoralis Major) were assessed at 0 and 5 days postmortem. A total of 216 female pheasant chicks were enrolled into the study. Twenty-four birds were euthanased at the start of the study, and samples of blood, breast muscle, leg muscle (M. Peroneus Longus and M. Gastrocnemius), heart, liver, kidney and gizzard were collected for determination of total Se. Remaining birds were blocked by live weight and randomly allocated to one of four dietary treatments (n = 48 birds/treatment) that either differed in Se source (SY v. SS) or dose (control (0.17 mg total Se/kg), SY-L and SS-L (0.3 mg/kg total Se as SY and SS, respectively) and SY-H (0.45 mg total Se/kg)). Following 42 and 91 days of treatment, 24 birds per treatment were euthanased, and samples of blood, breast muscle, leg muscle, heart, liver, kidney and gizzard were retained for determination of total Se and the proportion of total Se comprised as SeMet or SeCys. Whole blood GSH-Px activity was determined at each time point. Tissue GSH-Px activity and TBARS were determined in breast tissue at the end of the study. There were increases in both blood and tissues to the graded addition of SY to the diet (P < 0.001), but the same responses were not apparent with the blood and tissues of selenite-supplemented birds receiving a comparable dose (SY-L v. SS-L). Although there were differences between tissue types in the distribution of SeMet and SeCys, there were few differences between treatments. There were effects of treatment on erythrocyte GSH-Px activity (P = 0.012) with values being higher in treatments SY-H and SS-L when compared with the negative control and treatment SY-L. There were no effects of treatment on tissue GSH-Px activity, which is reflected in the overall lack of any treatment effects on TBARS.     

Size homogeneity of plasma prolactin in the female rat following various natural, pharmacological and stressful stimuli

Gel filtration on Sephadex G-150 was performed on freshly drawn plasma from ovariectomized, estrogen-treated rats following 10 minutes of ether inhalation, intraperitoneal administration of TRH (1 μg/rat) or pimozide (500 μg/kg body weight) and at the peak of the estrogen-induced afternoon surge of prolactin (1700 h). Plasma from intact lactating rats 30 minutes after suckling was also subjected to gel filtration. For comparison, homogenates of fresh and frozen pituitaries from ovariectomized, estrogen-treated rats were chromatographed. Prolactin activity was determined by RIA in each fraction eluted between the void and total volumes of the column. Immunoreactive prolactin in plasma following all experimental procedures eluted as a single component with a K_av of approximately 0.6. Chromatography of the fresh pituitary homogenate showed prolactin immunoactivity at the void volume and at a K_av of 0.4 and 0.6. A homogenate of frozen pituitary contained a component with a K_av of 0.3 in addition to those seen in fresh pituitary. These studies demonstrate that although the rat pituitary contains multiple molecular forms of immuno-reactive prolactin, only one small component is found in plasma.     

Selenium supplementation of lactating dairy cows: effects on milk production and total selenium content and speciation in blood, milk and cheese

Forty multiparous Holstein cows were used in a 16-week continuous design study to determine the effects of either selenium (Se) source, selenised yeast (SY) (derived from a specific strain of Saccharomyces cerevisiae CNCM I-3060) or sodium selenite (SS), or Se inclusion rate in the form of SY in the diets of lactating dairy cows on the Se concentration and speciation in blood, milk and cheese. Cows received ad libitum a total mixed ration (TMR) with a 1 : 1 forage : concentrate ratio on a dry matter (DM) basis. There were four diets (T1 to T4), which differed only in either source or dose of Se additive. Estimated total dietary Se for T1 (no supplement), T2 (SS), T3 (SY) and T4 (SY) was 0.16, 0.30, 0.30 and 0.45 mg/kg DM, respectively. Blood and milk samples were taken at 28-day intervals and at each time point there were positive linear effects of Se in the form of SY on the Se concentration in blood and milk. At day 112, blood and milk Se values for T1 to T4 were 177, 208, 248 and 279 ± 6.6 and 24, 38, 57 and 72 ± 3.7 ng/g fresh material, respectively, and indicate improved uptake and incorporation of Se from SY. In whole blood, selenocysteine (SeCys) was the main selenised amino acid and the concentration of selenomethionine (SeMet) increased with the increasing inclusion rate of SY. In milk, there were no marked treatment effects on the SeCys content, but Se source had a marked effect on the concentration of SeMet. At day 112, replacing SS (T2) with SY (T3) increased the SeMet concentration of milk from 36 to 111 ng Se/g and its concentration increased further to 157 ng Se/g dried sample as the inclusion rate of SY increased further (T4) to provide 0.45 mg Se/kg TMR. Neither Se source nor inclusion rate affected the keeping quality of milk. At day 112, milk from T1, T2 and T3 was made into a hard cheese and Se source had a marked effect on total Se and the concentration of total Se comprised as either SeMet or SeCys. Replacing SS (T2) with SY (T3) increased total Se, SeMet and SeCys content in cheese from 180 to 340 ng Se/g, 57 to 153 ng Se/g and 52 to 92 ng Se/g dried sample, respectively. The use of SY to produce food products with enhanced Se content as a means of meeting the Se requirements is discussed.     

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.     

Metabolism of selenomethionine and effects of interacting compounds by mammalian cells in culture

Since differences have been found in animals, the efficacies of selenomethionine (SeMet), selenite, and selenocystine (SeCys) for glutathione peroxidase (GPx) induction and cellular incorporation were compared and some effects of interacting nutrients on SeMet utilization were examined in tissue cultures. In three cell lines, Chang liver cells, mouse myoblasts and human fibroblasts, selenite was more effective than SeMet for GPx induction. However, radiotracer studies showed that SeMet was more rapidly incorporated into all cells than either selenite or SeCys. Chromatography of acid hydrolysates of Chang liver cells grown with 75Se-labeled SeMet indicated that approximately 90% of incorporated 75Se remained as SeMet, and less than 10% was as SeCys, the form of Se in GPx. Selenite supplementation slightly reduced both the incorporation of 75SeMet and the proportion of cellular 75Se recoverable as SeCys in Chang liver cells. Supplementation with L-methionine, however, significantly reduced 75SeMet incorporation, but significantly increased the proportion of cellular 75Se recovered as SeCys. L-cystine supplementation had no effect on either the cellular incorporation of 75SeMet or the proportion of cellular 75Se recovered as SeCys. These studies of SeMet utilization and effects of interacting nutrients are reflective of observations on SeMet metabolism in whole animals and humans.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000.     

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized α-subunits and from preformed cross-reacting protein

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.     

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes.

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

The major polypeptides of the murine-mammary-tumor virus isolated by plant-lectin affinity chromatography.

Solubilized polypeptides of the murine mammary tumor virus (MuMT virus) were chromatographed on a column of immobilised concanavalin A. The unbound viral material was rechromatographed on phosphocellulose, resulting in the isolation of the major proteins with a molecular weight of 28000 (p28) and 12000 (p12) respectively. The adsorbed glycopolypeptides after elution with methyl alpha-D-mannopyranoside were subjected to gel filtration. The major glycoprotein with a molecular weight of 52000 (gp52) was obtained in an almost pure form. However, a considerable part of gp52 elutes together with a glycoprotein with a molecular weight of 36000 (gp36), suggesting that in addition to the free form of gp52 a complex exists of gp52 plus gp36.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.