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R M Roberts

Publications and source records attributed to R M Roberts.

At least 163 records · Page 9Linked to original sources
PubMed1988

Aprotinin inhibits urokinase but not tissue-type plasminogen activator.

The protease inhibitor, aprotinin, has been examined for its ability to inhibit urokinase and tissue-type plasminogen activators at pH 7.4 in assays utilizing pyroGlu-Gly-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide as substrates, respectively. Aprotinin inhibited both two-chain low molecular weight urokinase and the high molecular weight form of the enzyme in a competitive manner with a similar Ki (27 microM). There was no observable inhibition of tissue-type plasminogen activators at aprotinin concentrations up to 500 microM. These findings suggest that sensitivity to inhibition by aprotinin could be used to distinguish tissue-type and urokinase-type plasminogen activators.

Aprotinin↗
PubMed1988

Conceptus development, uterine response, blood gases and endocrine function of gilts exposed to increased ambient temperature during early pregnancy.

Fifteen crossbred gilts were used to determine the influence of heat stress during Days 8 to 16 after onset of estrus on the development of conceptuses and uterine and endocrine functions. Ten gilts were bred 12 and 24 h after the onset of estrus (Day 0), and five gilts were nonbred controls. On Day 5, catheters were inserted into the uterine-ovarian vein (UV), saphenous artery (SA) and saphenous vein (SV) of each gilt. An electromagnetic blood flow transducer was implanted around the main uterine artery. Pregnant (n=5) and nonbred (n=5) control gilts were exposed to 21 +/- 1 degrees C, and pregnant heat-stressed gilts (n=5) were exposed to 37 +/- 1 degrees C for 12 h and 32 +/- 1 degrees C for 12 h daily during Days 8 through 16 after estrus. Treatment did not influence the partial pressure of oxygen (PO(2)) and of carbon dioxide (PCO(2)) in the UV, SA and SV blood. Uterine blood flow was not altered by heat stress. On Day 16, total wet weight of conceptuses was reduced in the gilts that were heat-stressed compared with conceptuses from control gilts. Incorporation of (3)H-leucine into macromolecules in vitro by conceptuses from the heat-stressed gilts was reduced compared with control gilts. Concentrations of 15-keto-13, 14-dihydro prostaglandin F(2alpha) (PGFM) in peripheral blood were greater than 1 ng/ml between Days 13 to 16 after estrus in 20% of the pregnant control gilts, 60% of the heat-stressed pregnant gilts, and 100% of the nonbred gilts. Concentrations of estradiol in the SA were affected by treatment. These results indicate that heat stress of gilts between Days 8 to 16 after estrus reduced the amount of conceptus tissue and altered concentrations of estradiol in the peripheral circulation, but uterine blood flow and PO(2) and PCO(2) in blood were not affected.

Journal Article↗
PubMed1988

Ovine trophoblast protein-1 and bovine trophoblast protein-1 are present as specific components of uterine flushings of pregnant ewes and cows.

A rabbit antiserum raised against ovine trophoblast protein-1 (oTP-1) was used to stain Western blots of the protein components from the uterine flushings of pregnant ewes (n = 61), non-bred cyclic ewes (n = 22), bred-but-nonpregnant ewes (n = 36), pregnant cows (n = 34), and bred-but-nonpregnant cows (n = 15). Nonpregnant animals were defined as ones from which no embryo was recovered. Uterine flushings of pregnant ewes contained oTP-1 between Days 14 and 24 of pregnancy, but not at Day 12. All of the cyclic ewes and 34 of 36 bred ewes, judged as nonpregnant, tested negatively for the presence of oTP-1. With one exception, oTP-1 was not detected in the nongravid uterine horns of pregnant ewes in which the conceptus had been confined to one uterine horn. Bovine trophoblast protein-1 (bTP-1), which cross-reacts immunologically with oTP-1, was also detectable specifically in the uterine flushings of pregnant cows when anti-oTP-1 antiserum was used. The urine (n = 14) and certival mucus (n = 20) samples of all the pregnant ewes tested were free of any detectable oTP-1. Thus, a useful pregnancy test for ewes based on oTP-1 release into these fluids seems unlikely. Results of this study show that oTP-1 and bTP-1 are pregnancy-specific proteins that are secreted into the uterine lumen where they possibly exert a local response.

Animals↗
PubMed1988

Synthesis and processing of ovine trophoblast protein-1 and bovine trophoblast protein-1, conceptus secretory proteins involved in the maternal recognition of pregnancy.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) are newly discovered proteins produced by embryonic tissues for a limited period in early gestation. They appear to act as agents that prevent regression of the corpus luteum during early pregnancy in the ewe and cow. Ovine TP-1 [mol wt (Mr), 17,000] consists of three or four isoelectric variants (pI 5.4-5.7), whereas bTP-1, which cross-reacts with antiserum to oTP-1, is found as two predominant Mr classes (Mr, 22,000 and 24,000), each with several isoelectric variants (in the pI range 6.3-6.8). Cell-free translation of ovine conceptus mRNA yields pre-oTP-1 also consists of three or four isoelectric variants, assumed to have arisen by translation of multiple mRNA species. Ovine TP-1 is not glycosylated. When bovine conceptus mRNA is translated, a group of four or five isoforms of pre-bTP-1 are generated, each with a Mr of 19,000. In the presence of microsomes the Mr shifts upward to about 21,500. Bovine conceptuses cultured in presence of either [3H]glucosamine or [3H]mannose incorporate label into both size classes of bTP-1 (Mr, 22,000 and 24,000). Culture in presence of [35S]methionine and tunicamycin gave rise to a nonglycosylated form of bTP-1 with an apparent Mr of 18,000. Treatment of [35S]methionine-labeled bTP-1 with either endoglycosidase-H or peptide:N-glycosidase F yielded products with Mr of 17,000 and 16,000, respectively. bTP-1, although functionally and structurally related to oTP-1, appears to be a glycoprotein carrying at least two Asn-linked oligosaccharides. The two Mr classes of bTP-1 arise as a result of differences in either the number or structure of the carbohydrate chains. Like oTP-1, bTP-1 is probably translated from multiple mRNA species.

Animals↗
PubMed1988

Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein.

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.

Acid Phosphatase↗
PubMed1988

The functions of uterine secretions.

The likely functions of uterine secretions, often termed histotroph, in the nurture of the early conceptus are reviewed. Particular emphasis has been placed on the pig in which the uterus synthesizes and secretes large amounts of protein in response to progesterone. In this species, which possesses a non-invasive, diffuse type of epitheliochorial placentation, the secretions provide a sustained embryotrophic environment which is distinct from that of serum. A group of basic proteins dominates these uterine secretions after Day 11 of pregnancy and its best characterized member is uteroferrin, an iron-containing acid phosphatase with a deep purple colour. Evidence has accumulated to suggest that uteroferrin, rather than functioning as an acid phosphatase, is involved in transporting iron to the conceptus. Three basic polypeptides which are found noncovalently associated with uteroferrin have been shown to be antigenically closely related to one another and to have arisen by post-translational processing from a common precursor molecule. Their function is unknown. A group of basic protease inhibitors has been identified which bear considerable sequence homology to bovine pancreatic trypsin inhibitor (aprotinin) and may control intrauterine proteolytic events initiated by the conceptuses. The last basic protein so far characterized is lysozyme which is presumed to have an antibacterial role. Finally, two low molecular weight (Mr approximately 18,000) acidic polypeptides have been purified and have sequence homology to a plasma retinol binding protein. Like uteroferrin, these proteins may be responsible for transport of an essential nutrient to the conceptus.

Animals↗
PubMed1987

Sequence homology in the metalloproteins; purple acid phosphatase from beef spleen and uteroferrin from porcine uterus.

The primary structures of purple acid phosphatase and uteroferrin, two iron-binding glycoproteins isolated from beef spleen and porcine uterine fluids, respectively, have been examined by a combination of tandem mass spectrometry and classical Edman sequencing methods. Reported here are amino acid sequence data covering more than 90% of the primary structures for these two proteins. The sequence data reveal an unexpectedly high degree of homology, greater than 90%, for these two proteins.

Acid Phosphatase↗
PubMed1987

Hormonal control and function of secretory proteins.

The uterus of the pig secretes large amounts of protein in response to progesterone. Estrogen alone has little effect but in combination with progesterone is synergistic at low doses and inhibitory at high doses. The responses of the uterus to progesterone require prolonged hormone treatment and are not immediate. The proteins secreted by the uterus of all species are believed to play some role in the nutritional and developmental support of the conceptuses, particularly during early pregnancy. Such a role is likely to be of greater importance in species such as the pig which possesses a noninvasive, diffuse-type of epitheliochorial placentation. A group of basic polypeptides dominates the uterine secretions of the pig. The best characterized is uteroferrin, a purple colored, iron-containing acid phosphatase which transports iron across the placenta. Three polypeptides which are found associated noncovalently with uteroferrin have been shown to be antigenically closely related to each other and to have arisen from a single precursor polypeptide. Their function is unknown. A family of plasmin/trypsin inhibitors which show sequence homology with bovine pancreatic trypsin inhibitor (aprotinin) has been well characterized and appears to control intrauterine proteolytic events initiated by the conceptuses. Several other proteins secreted in response to progesterone remain to be characterized and functionally defined.

Acid Phosphatase↗
PubMed1987

Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus.

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.

Amino Acid Sequence↗
PubMed1987

Purification, secretion and immunocytochemical localization of the uterine milk proteins, major progesterone-induced proteins in uterine secretions of the sheep.

Restriction of the conceptus to one uterine horn of the pregnant ewe results in the accumulation of fluid called uterine milk (UTM) in the contralateral horn. Two basic polypeptides, called the uterine milk proteins (UTM-proteins; Mr = 55,000 and 57,000 as determined by polyacrylamide-gel electrophoresis using sodium dodecyl sulfate), accounted for the majority of the protein in uterine milk. The two UTM-proteins were glycoproteins and were readily purified from uterine fluids by cation-exchange chromatography on carboxymethyl (CM)-cellulose followed by Sephacryl S-200 gel-filtration. The purified UIM-proteins had a weight-average molecular weight of 50,700 +/- 4,200, as determined by equilibrium sedimentation analysis. Endometrial explants from pregnant ewes were cultured in the presence of radioactive amino acids and released UTM-proteins into the medium as their major secretory products. The UTM-proteins were secreted into the uterine lumen of nonpregnant, ovariectomized ewes given daily injections of progesterone. Estrone alone was ineffective in inducing UTM-protein production. Immunocytochemical studies indicated that synthesis of the UTM-proteins was confined to the surface and glandular epithelium of the uterus.

Animals↗
PubMed1987

The effect of ovine trophoblast protein-one on endometrial protein secretion and cyclic nucleotides.

The effect of ovine trophoblast protein-one (oTP-1) on endometrial protein secretion was examined by using a dual radioisotope technique in which 3H- and 35S-methionine were employed to measure relative rates of protein release into the medium by endometrial explant cultures (Exp. I). Endometrium (200 mg) from Day (D) 12 of the cycle was cultured with either 5 micrograms/ml oTP-1, 5 micrograms/ml bovine serum albumin (BSA) or 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (DbcAMP). Culture media from control BSA and treated explant cultures were mixed. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and detected by fluorography. Individual protein spots were punched from gels, extracted, and their radioactive content measured. Ratios of 3H:35S were used to determine treatment effects. In Experiment II, 3H- and 14C-leucine were used for the dual radiolabel, and the DbcAMP treatment was omitted. In both experiments, a protein having a molecular weight (Mr) of about 70,000 and a pI approximately equal to 4 was increased (p less than 0.01) 200-400% by oTP-1. Secretion of several other endometrial proteins was also amplified in the presence of oTP-1. The polypeptides that increased in response to oTP-1 were inhibited by DbcAMP, and vica versa. In Experiment III, endometrial explants from D12 cyclic ewes were cultured for 4 h with either 5 micrograms/ml oTP-1 or 5 micrograms/ml BSA to determine whether oTP-1 influenced concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Concentrations of cAMP in oTP-1-treated endometrium were lower (p less than 0.1) than in BSA-treated endometrium (0.29 vs. 0.41 pmoles/mg tissue, respectively). Levels of cGMP were unaffected by oTP-1. In Experiment IV, endometrium from D14 of the cycle was incubated in medium alone or in medium containing either 2 micrograms/ml oTP-1, 1 microgram/ml oxytocin (OXY), or oTP-1-plus-OXY. None of the treatments significantly affected cAMP levels. In Experiment V, D16 endometrium was collected from pregnant and nonpregnant ewes that had received either 0 or 10 IU OXY i.v. cAMP was higher (p less than 0.01) in endometrium from pregnant ewes compared to nonpregnant ewes (27.9 vs. 13.0 pmoles/mg tissue, respectively), but OXY had no detectable effect on endometrial content of cAMP in either nonpregnant or pregnant ewes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Suppression of lymphocyte activation by a high-molecular-weight glycoprotein released from preimplantation ovine and porcine conceptuses.

A high-molecular-weight (MW greater than 660,000), acidic glycoprotein (HMWGP) was purified from incubation medium of preimplantation, elongating ovine (day 16 and 17) and porcine (day 16) conceptuses. HMWGP was tested for its ability to inhibit [3H-methyl]thymidine incorporation into lymphocytes stimulated by phytohemagglutinin or two-way mixed lymphocyte cultures. Ovine and porcine HMWGP inhibited the incorporation of [3H-methyl]thymidine into lymphocytes in a dose-dependent manner. An approximately 50% inhibition was detected at the lowest dose tested (ovine, 25 micrograms/ml; porcine, 5 micrograms/ml). Complete suppression of thymidine incorporation occurred at the highest doses evaluated (ovine, 200 micrograms/ml; porcine, 40 micrograms/ml). This immunosuppressive effect was not the result of an overall cytotoxic effect on lymphocytes as evaluated by trypan blue exclusion. In conclusion, an ovine and porcine conceptus glycoprotein, HMWGP, has potent in vitro immunosuppressive activity in both phytohemagglutinin and mixed lymphocyte cultures. In vivo, HMWGP may have an immunoregulatory role during early pregnancy in the sheep and pig by providing a local immunosuppressive environment within the uterus to prevent conceptus rejection.

Animals↗
PubMed1987

Effects of early pregnancy and acute 17 beta-estradiol administration on porcine uterine secretion, cyclic nucleotides, and catecholamines.

This study investigated acute effects of 17 beta-estradiol (E) on ions, cyclic nucleotides, and catecholamines and their association with temporal changes in uterine secretory products in pregnant, cyclic, and nonpregnant gilts. Uterine flushings (UTF) and endometrium (ENDOM) from one uterine horn of nonpregnant and pregnant gilts (n = 9) were collected on days 10, 12, and 14 (n = 3). Protein, plasma inhibitor (P less than 0.05), Na+, and K+ (P less than 0.01) increased linearly in UTF of pregnant gilts. Ca2+ changed biphasically, with higher concentrations (P less than 0.01) in pregnant gilts on day 12. Endometrial cAMP and cGMP (P less than 0.05) increased between days 12 and 14 of pregnancy. The UTF norepinephrine (NE) concentrations increased (P less than 0.01) in cyclic gilts between days 12 and 14, while endometrial NE increased between days 10 and 12 and then decreased on day 14. The UTF of pregnant gilts had higher (P less than 0.05) concentrations of dopamine (DA), which peaked on day 12 and then decreased (P less than 0.01) by day 14. DA in UTF of nonpregnant gilts decreased between days 10 and 12 and remained low on day 14. A catecholamine metabolite, 3,4-dihydroxyphenylglycol, in ENDOM (P less than 0.05) and UTF (P less than 0.01) decreased linearly between days 10 and 14. The turnover rate of endometrial catecholamines was 2.4-fold higher (P less than 0.05) during early pregnancy compared with that in cyclic gilts. Effects of acute administration of exogenous E (0.5 mg) were also studied. The UTF and ENDOM were from day 11 nonpregnant gilts 0, 30, 60, and 360 min (n = 3) post-E or post-saline-ethanol (C) injection (n = 9). Potassium, plasma inhibitor (P less than 0.01), and cGMP (P less than 0.05) increased rapidly (30 min) after E injection. The K+ level changed biphasically, with increased concentrations again at 360 min. Plasmin inhibitor returned to levels similar to controls by 60 min, whereas cGMP remained elevated until 360 min postinjection when E, Ca2+, K+ (P less than 0.01), and Na+ (P less than 0.03) were increased. ENDOM NE (P less than 0.05) and UTF DA (P less than 0.05) were lower in E-treated gilts. Additionally, the turnover rate was significantly lower (P less than 0.05) for NE and DA in the UTF of E-treated gilts. An increase in Ca2+ in UTF preceded increases in protein secretions (except plasmin inhibitor) and may play a role in E-stimulated endometrial epithelial secretory vesicle exocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1987

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1.

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.

Animals↗
PubMed1987

Secretory function of the ovine uterus: effects of gestation and steroid replacement therapy.

In Exp. 1, changes in uterine fluid content of protein, calcium and prostaglandin F2 alpha (PGF) were examined between d 30 to 144 of gestation. Volume of uterine fluid (mean +/- SE) in the nongravid uterine horn of unilaterally pregnant ewes increased (P less than .05) between d 30 (8 +/- 1 ml) and d 144 (749 +/- 46 ml) of gestation. Protein concentration and total protein in uterine fluid also increased (P less than .05) between d 30 (2.3 +/- .4 mg/ml; 19 +/- 7 mg) and d 144 (23.5 +/- 3.3 mg/ml; 17.4 +/- 2.0 g). Total recoverable calcium (mg) in secretions increased from .1 mg (d 30) to over 1.4 g (d 144) due to day of gestation effects (P less than .05). Total PGF also increased (P less than .01) from 7 ng on d 30 to 15.7 g on d 144. In Exp. II, ovariectomized (OVX) ewes were treated with either corn oil, estrone (E), progesterone (P) or P+E (PE) for 30 d. Treatment with P or PE increased calcium concentration and content (P less than .01) in secretions, but differences in uterine fluid volumes and concentrations of protein and PGF were not significant. Treatment of OVX ewes with P stimulated (P less than .05) in vitro synthesis of secretory proteins by endometrium, whereas treatment with E enhanced release of unlabeled proteins (P less than .05). The major endometrial secretory proteins were identified in allantoic fluids from d 60 to term and were detected along the mesenchymal border of the chorioallantois using immunohistochemistry. Results from this study indicate that P may be a primary hormone regulating accumulation of fluid, protein, calcium and specific endometrial proteins in the uterine lumen during gestation, and that uterine milk proteins gain access to the fetal-placental unit.

Animals↗
PubMed1986

Isolation and characterization of a high molecular weight stable pink form of uteroferrin from uterine secretions and allantoic fluid of pigs.

A pink, high molecular weight form of uteroferrin (Uf) has been isolated from uterine secretions and allantoic fluid of pigs. This protein fraction (denoted FIII) which is relatively stable under physiological conditions of pH, ionic strength, and temperature has a molecular weight of about 80,000, a value approximately twice that of purple Uf (Mr approximately 35,000) isolated from a separate fraction (FIV) by gel filtration. The visible absorption spectrum, EPR signal, and acid phosphatase activity of Uf in FIII are almost identical to those of FIV Uf after the latter has been reduced by 2-mercaptoethanol. However, unlike reduced FIV Uf, the pink, high molecular form does not revert to purple, nor does it show loss of EPR signal and phosphatase activity in the presence of oxygen. In addition, it does not become purple at orthophosphate concentrations which inhibit Uf acid phosphatase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has shown that FIII consists of approximately equal amounts of Uf polypeptides (Mr = 35,000 and 37,000) and a group of three polypeptides (Mr = 40,000, 46,000, and 50,000) antigenically unrelated to Uf. The latter share a common epitope not found on Uf and are probably differentially processed forms of the same protein. FIII can be dissociated by pH conditions below 5.0, by exposure to antibodies raised against Uf or the associated polypeptides, and by sodium dodecyl sulfate at 100 degrees C. The polypeptides in FIII are not therefore linked by disulfide bonds. Treatment with dimethyl suberimidate, however, results in a cross-linked complex (Mr approximately 82,000) consisting of Uf and the associated polypeptides. It is concluded that this high Mr form of Uf is a heterodimer of fully activated Uf and a second polypeptide of unknown function.

Acid Phosphatase↗
PubMed1986

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues.

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.

Acid Phosphatase↗