Purificação, caracterização e dosagem radioimunológica de glicopoteínas associadas à gestação em zebuínos

Abstract

The pregnancy associated glycoproteins (PAGs) constitute a large family of aspartic pro teinases expressed in the outer epithelial cell layer of the Artyodactyla placenta. In the first part of the present work, two biochemical approaches were used to characterize PAGs isolated from zebu ( Bos indicus ) fetal cotyledons. The first procedure, use d to isolate PAG from zebu placenta removed late in pregnancy, included extraction of proteins at neutral pH, acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies. The second procedure, used to investigate PAG in placentas removed at early and mid gestational periods, included protein extractions at acid or alkaline pH followed by pepstatin A agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation proc edures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS PAGE and Western Blot, before transfer to polyvinylidene difluoride (PVDF) membrane for NH 2 microsequence determination. By use of SDS PAGE and Western Blot, different isoforms of PAG with apparent molecular masses from 51 to 69 kDa and isoeletric points varying from 3.1 to 6.7 were identified in placentas from different gestational ages . After CM ceramic chromatography of all except 0.32 M NaCl DEAE fraction, the most immunoreactive proteins revealed N terminal amino acid sequences (10 to 25 aa long) which were 100% identical to bovine PAG 1. The same sequence (14 aa long) was found after pepstatin agarose affinity chromatography of prote ins extracted from placentas removed earlier in pregnancy. These results converged towards the expression of one major N terminal PAG amino acid sequence in zebu placentas at different gestational ages. In the second part of this study, two specific RIA sy stems were developed then used to measure plasmatic PAG concentrations during gestation and postpartum period in Azawak zebu cows. Twelve females palpated per rectum and diagnosed as pregnant were bled at 5 10 days interval approximately from Week 8 of ges tation till Week 10 postpartum (pp). One zebu cow initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow showed abnormally high PAG concentrations during gestation , being excl uded from the general PAG profile. The 10 other zebu cows gave a very homogeneous PAG profile. In these animals, concentrations increased progressively from Week 8 to Week 35 of gestation (from 6.0  4.2 to 196.0  34.8 ng/ml), remaining relatively constant un til Week 39 (210.8  74.8 ng/ml), when they increased sharply to reach their highest level (1,095.6  607.2 ng/ml) at parturition. After delivery, PAG concentrations declined significantly (P<0.05) till the Week 2 postpartum (348.4  85.6 ng/ml) and slowly till the Week 10 postpartum . These results revealed that PAG concentrations in zebu cattle were lower than those previously described in taurine breeds between week 35 and 39 of gestation