[en] In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.
Afting, E.G., Becker, M.L., Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield (1981) Biochem. J, 197, pp. 519-522
Butler, J.E., Hamilton, W.C., Sasser, R.G., Ruder, C.A., Hass, G.M., Williams, R.J., Detection and partial characterization of two bovine pregnancy-specific proteins (1982) Biol. Reprod, 26, pp. 925-933
Camous, S., Charpigny, G., Guillomot, M., Martal, J., Purification of one pregnancy-specific protein by high-performance liquid chromatography (HPLC) (1988), Proceedings of Bard Workshop, Maternal Recognition of Pregnancy and Maintenance of the Corpus Luteum, Jerusalem Abstract 2
Davis, M.O., Hata, D.J., Smith, D., Walker, J.C., Cloning and sequence of a chicken alpha-N-acetylgalactosaminidase gene (1993) Biochim. Biophys. Acta, pp. 296-298. , 1216
Dzau, V.J., Slater, E.E., Haber, E., Complete purification of dog renal renin (1979) Biochemistry, 18, pp. 5224-5228
Garbayo, J.M., Remy, B., Alabart, J.L., Folch, J., Wattiez, R., Falmagne, P., Beckers, J.F., Isolation and partial characterization of a pregnancy-associated glycoprotein family from the goat placenta (1998) Biol. Reprod, 58, pp. 109-115
Garbayo, J.M., Green, J.A., Manikkam, M., Beckers, J.F., Kiesling, D.O., Ealy, A.D., Roberts, R.M., Caprine pregnancy-associated glycoproteins (PAG): Their cloning, expression, and evolutionary relationship to other PAG (2000) Mol. Reprod. Dev, 57, pp. 311-322
Green, J.A., Xie, S., Quan, X., Bao, B., Gan, X., Mathialagan, N., Beckers, J.F., Roberts, R.M., Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy (2000) Biol. Reprod, 62, pp. 1624-1631
Guruprasad, K., Blundell, T.L., Xie, S., Green, J., Szafranska, B., Nagel, R.J., McDowell, K., Roberts, R.M., Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases (1996) Protein Eng, 9, pp. 849-856
Herrmann, T., Schindler, D., Tabe, H., Onodera, O., Igarashi, S., Polack, A., Zehnpfennig, D., Tsuji, S., Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene (1998) Gene, 211, pp. 205-214
Huang, F., Cockrell, D.C., Stephenson, T.R., Noyes, J.H., Sasser, R.G., Isolation, purification, and characterization of pregnancy-specific protein B from elk and moose placenta (1999) Biol. Reprod, 61, pp. 1056-1061
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.G., Protein measurement with the folin phenol reagent (1951) J. Biol. Chem, 193, pp. 265-275
Lynch, K.A., Alexander, R.M., Sasser, R.G., The cloning and expression of the bovine pregnancy specific protein B (bPSPB) gene (1992) Biol. Reprod, 46 (SUPPL. 1), p. 73. , (Abstract 89)
Mialon, M.M., Camous, S., Renand, G., Martal, J., Menissier, F., Peripheral concentrations of a 60-kDa pregnancy serum protein during gestation and after calving and in relationship to embryonic mortality in cattle (1993) Reprod. Nutr. Dev, 33, pp. 269-282
Nierman, W.C., Feldblyum, T.V., Laub, M.T., Paulsen, I.T., Nelson, K.E., Eisen, J., Heidelberg, J.F., Fraser, C.M., Complete genome sequence of Caulobacter crescentus (2001) Proc. Natl. Acad. Sci. USA, 98, pp. 4136-4141
Pearson, W.R., Lipman, D.J., Improved tools for biological sequence (1988) Proc. Natl. Acad. Sci. USA, 85, pp. 2444-2448
Sasser, R.G., Ruder, C.A., Ivani, K.A., Butler, J.E., Hamilton, W.C., Detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation (1986) Biol. Reprod, 35, pp. 936-942
Sousa, N.M., Figueiredo, J.R., Beckers, J.F., Placental proteins in ruminants: Biochemical, physiological and zootechnical aspects (2001) Biotechnology in Animal Husbandry, pp. 179-208. , Renaville R., Burny A. (Eds.), 1st ed., Dordrecht: Kluwer Academic Publishers
Sweeley, C.C., Ledonne N.C., Jr., Robbins, P.W., Post-translational processing reactions involved in the biosynthesis of lysosomal alpha-N-acetylgalactosaminidase in cultured human fibroblasts (1983) Arch. Biochem. Biophys, 223, pp. 158-165
Towbin, H., Stachelin, T., Gordon, J., Eletrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications (1979) Proc. Natl. Acad. Sci. USA, 76, pp. 4350-4354
Tsuji, S., Yamauchi, T., Hiraiwa, M., Isobe, T., Okuyama, T., Sakimura, K., Takahashi, Y., Miyatake, T., Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B) (1989) Biochem. Biophys. Res. Commun, 163, pp. 1498-1504
Wang, A.M., Bishop, D.F., Desnick, R.J., Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene (1990) J. Biol. Chem, 265, pp. 21859-21866
Willard, J.M., White, D.R., Wesson, C.A., Stellflug, J., Sasser, R.G., Detection of fetal twins in sheep using a radioimmunoassay for pregnancy-specific protein B (1995) J. Anim. Sci, 73, pp. 960-966
Xie, S., Low, R.C., Nagel, R.J., Kramer, K.K., Anthony, R.V., Zoli, A.P., Beckers, J.F., Roberts, R.M., Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family (1991) Proc. Natl. Acad. Sci. USA, 88, pp. 10247-10251
Xie, S., Green, J.A., Bao, B., Beckers, J.F., Valdez, K.E., Hakami, L., Roberts, R.M., Multiple pregnancy-associated glycoproteins are secreted by day 100 ovine placental tissue (1997) Biol. Reprod, 57, pp. 1384-1393
Xie, S., Green, J.A., Bixby, J.B., Szafranska, B., DeMartini, J.C., Hecht, S., Roberts, R.M., The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes (1997) Proc. Natl. Acad. Sci. USA, 94, pp. 12809-12816
Zoli, A.P., Beckers, J.F., Wouters-Ballman, P., Closset, J., Falmagne, P., Ectors, F., Purification and characterization of a bovine pregnancy-associated glycoprotein (1991) Biol. Reprod, 45, pp. 1-10
Zoli, A.P., Guilbault, L.A., Delahaut, P., Benitez-Ortiz, W., Beckers, J.F., Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: Its application in pregnancy diagnosis (1992) Biol. Reprod, 46, pp. 83-92