Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10⁶-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.