Structural analysis of acute-phase alpha 2-macroglobulin.

High resolution images of rat acute-phase alpha 2-macroglobulin (AP alpha 2M) have been obtained by using dark-field electron microscopy. No staining or artifact-inducing procedures were used. Analysis of unfiltered electron microscope plates, exposed to minimal electron beam radiation, revealed highly contrasted particles of variable morphology with dimensions of approx. 19 nm X 14 nm. An electron-dense core with four to six projections could be seen. Two-fold symmetry was evident in selected images, supporting the four-subunit composition of the protein. Image processing and filtering confirmed the presence and configuration of the projections by demonstrating exact molecular dimensions of 16 nm X 9.5 nm and a shape with six projections like that of the Russian letter zh. SDS/polyacrylamide-gel electrophoresis revealed that this molecule was in the proteinase-bound form. C.d. data revealed a surprisingly low content of alpha-helical secondary structure (12%) and an atypically large content of beta-form structure (33%). Comparison of the amino acid compositions of AP alpha 2M and human alpha 2-macroglobulin indicated a high degree of homology between the two molecules. It is concluded that the conformation of rat AP alpha 2M, both at the molecular and secondary structural levels, is strikingly similar to that of human alpha 2-macroglobulin.

Distribution of acute-phase alpha 2-macroglobulin in rat fetomaternal compartments.

Rat acute-phase alpha 2-macroglobulin (AP alpha 2M) concentration was measured by radioimmunoassay in maternal serum, fetal plasma, maternal liver, fetal liver, and amniotic fluid as a function of gestational and neonatal age. The concentration profiles of AP alpha 2M in maternal serum and fetal plasma displayed two peaks, one in early gestation and another during late gestation. Synthesis of AP alpha 2M was confirmed by the immunoprecipitation of [35S]methionine incorporated into cultures of selected tissues. The following observations were made. 1) Maternal serum concentrations of AP alpha 2M were higher than those observed in fetal plasma in early gestation. This was attributable to a high level of maternal AP alpha 2M synthesis in metrial gland which was absent in liver and moderate in yolk sac. 2) In late gestation fetal plasma concentrations of AP alpha 2M greatly exceeded those observed in maternal serum. This could be explained by the pronounced synthesis of AP alpha 2M in fetal liver that was not apparent in maternal liver or yolk sac. 3) During labor, a transient increase in AP alpha 2M concentration was observed in maternal serum and fetal plasma. 4) During lactation a moderately elevated maternal serum concentration of AP alpha 2M was maintained. 5) Amniotic fluid concentration of AP alpha 2M was very low throughout gestation, which indicated that the fetal glomerulus was relatively impermeable to this large protein. It is concluded that in early gestation a principal maternal source of AP alpha 2M appears to be the metrial gland, whereas in late gestation fetal liver is a major source of AP alpha 2M appearing in fetal plasma from where some of this macroglobulin is speculated to be transported to the maternal circulation.

Synthesis of acute-phase alpha 2-macroglobulin during inflammation and pregnancy.

A recent investigation of acute-phase alpha 2-macroglobulin (AP alpha 2M) concentration in the rat during pregnancy demonstrated a bimodal distribution, for which we suggested a maternal source of AP alpha 2M in early gestation and a fetal source in late gestation. This interpretation was supported by the findings of the present study, which employed organ culture techniques, incorporation of [35S]methionine, immunoprecipitation of radioactivity, and fluorography to measure synthesis of AP alpha 2M in specific fetal, adult, and maternal tissues. Preliminary results indicated that in adult male rats treated with croton oil (compared with nontreated males), AP alpha 2M was synthesized in kidney, spleen, thymus, and lymphocytes by 48 hr post induction, but synthesis in the liver was not evident. In the pregnant rat from 12 to 16 days (compared with nonpregnant females), synthesis of AP alpha 2M was high in metrial gland, moderate in spleen, thymus and lymphocytes, and absent in liver; at 21 days, synthesis of AP alpha 2M in these four maternal tissues had declined. Fetal synthesis of AP alpha 2M in yolk sac (12 to 16 days) and in liver (15 to 16 days) was significantly elevated, and at 21 days fetal liver still displayed marked synthesis. These data are consistent with the interpretation that an early maternal source of AP alpha 2M synthesis is the metrial gland and that in the fetus both yolk sac and liver are major sources of AP alpha 2M, the latter tissue continuing synthesis into late gestation. Lymphopoietic and lymph-containing tissues appear to be major sites of AP alpha 2M synthesis during inflammation and pregnancy.

Acute-phase α2-macroglobulin in csf during development of the fetal rat.

The concentration of acute phase α2-macroglobulin (APα2M) was measured in the cerebrospinal fluid (csf) and plasma of fetal (12-22 days gestation) and neonatal (0-10 days post partum) rats. APα2M was detectable in the fetus as early as samples could be obtained (12 days) and increased in both fluids to reach a peak near the time of birth (17 mg/100 ml in csf and 168 mg/100 ml in plasma). During the neonatal period APα2M concentration declined markedly in both fluids. The results are compared with values for albumin and α-fetoprotein in fetal rats. It was concluded that maintenance of the csf:plasma ratios for the three proteins are incompatible with an explanation of passive diffusion from plasma to csf. Other mechanisms to explain the occurrence of high concentrations of plasma proteins in fetal csf are discussed.

Isolation and purification of rat acute-phase alpha 2-macroglobulin.

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.

Radioimmunoassay of rat acute-phase alpha 2-macroglobulin.

A double-antibody radioimmunoassay (RIA) to acute-phase alpha 2-macroglobulin was developed for the quantitation of this large macromolecule in physiological fluids. The primary receptor for the RIA was a monospecific antiserum to purified acute-phase alpha 2-macroglobulin which produced a high titre (7.5 . 10(6)) antibody with a strong affinity for rat acute-phase alpha 2-macroglobulin (Ka = 1.24 . 10(11)) as measured by Scatchard analysis. The validity of the assay was confirmed by specificity for rat alpha 2-macroglobulin measured in various physiological fluids as assessed by parallel dose-response curves; and accuracy, measured by the analytical recovery of alpha 2-macroglobulin by the RIA in serum (104 +/- 7%) and buffer (103 +/- 7%), and the correlation (R = 0.999) of measurements of acute-phase alpha 2-macroglobulin-containing samples measured in serum and buffer. Reference acute-phase serum measured by this RIA and by rocket immunoelectrophoresis were 98.6% in agreement. Radioimmunoassay sensitivity was estimated at less than 1.0 ng alpha 2-macroglobulin/ml, measured over a range of 0-160 ng. Precision was assessed by intraassay (2.99 +/- 0.97%) and interassay (8.76 +/- 2.64%) variation. Evaluation confirmed that quantitation of rat acute-phase alpha 2-macroglobulin by this RIA met the criteria of sensitivity, validity and precision.

Synthesis and secretion of alpha-fetoprotein and acute-phase alpha 2-macroglobulin by Zajdela rat ascites hepatoma.

Zajdela rat ascites hepatoma cells have been reported to be 'nonsecretors' of alpha-fetoprotein (AFP). Because of the potential of such cells in the study of defective secretory mechanisms of AFP and other serum proteins, we concluded a detailed investigation of the production of AFP in Zajdela cells growing in vivo. Three approaches were used: radioimmunoassay of intracellular and extracellular AFP and assays of AFP mRNA by molecular hybridization and cell-free translation. Radioimmunoassay showed that the AFP concentration in the hepatoma cells was extremely low (1.2 micrograms/g) as compared to known AFP-producing tissues such as fetal liver (600 micrograms/g). The assays of AFP mRNA by hybridization and translation supported the low level of AFP production in Zajdela cells, and we consider such cells to be poor producers of AFP and therefore not suitable for the study of defective mechanisms for AFP secretion. The Zajdela tumor cell intracellular concentration of acute-phase alpha 2-macroglobulin (AP alpha 2M) measured by radioimmunoassay was also found to be low (3.0 micrograms/g) compared to liver of Zajdela-bearing rats (80 micrograms/g). In view of these low levels of synthesis of AFP and AP alpha 2M in Zajdela hepatoma cells, little biological significance may be attached to the apparent nonsecretion of these onco-fetal proteins.

Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development.

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.