Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 1251-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal mucle myosin chymotryptic or Mg2+/ papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-I heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50 -100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: (a) it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and (b) it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-I heavy chain of about 100 amino acid residues.

Transforming growth factor-: (TGF-f) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-41 and TGF-f2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGFf1 and TGF-,32. The Kd values obtained from Scatchard analyses were -65 pM for 125I1 TGF-31 and -40 pM for 1251-TGF-fl2, with 70 000 and 85 000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-f1 and TGF-32 were equipotent (apparent half maximal inhibition [IC50] -70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 1251-TGF-l1 and 1251-TGF-32 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-f binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-31 than for -f2 yet exhibits a 5-to 10-fold higher affinity for TGF-,B2 than for -41. The 38-kDa TGF-1 binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-,B2 than for -f1 that is strikingly similar to that of the type III/ betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.

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Tubulin and actin are cytoskeletal proteins known to play a major role in dividing cells. Tetrahymena pyriformis, a ciliated protozoan, was used as a model system for investigating tubulin synthesis during cilia regeneration and during the cell cycle. Until recently the identification of actin in Tetrahymena has been controversial. In this report evidence for the presence of actin in Tetrahymena is reviewed and control of actin gene expression during the cell cycle is discussed.

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A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine. HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8–12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts.