Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids.

Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin. Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.

Characterization by affinity electrophoresis of an alpha-1,6-glucan-binding protein from Streptococcus sobrinus.

Glucan-binding protein 1 (GBP1), the most abundant glucan-binding protein isolated from culture supernatants of Streptococcus sobrinus 6715-49, has been purified by affinity chromatography on Sephadex G-50 followed by gel permeation chromatography with Bio-Gel P-10. The specificity and affinity of GBP1 for glucans were assessed by affinity electrophoresis. GBP1 did not detectably bind to glucans lacking linear arrays of alpha-1,6 linkages. The association constant for the linear alpha-1,6-glucan Dextran T2000 was 3 x 10(7) M-1. Providing small isomaltosaccharide ligands to compete with this dextran indicated that the binding site maximally accommodated isomaltosaccharides with a degree of polymerization of 8. When glucans produced by purified S. sobrinus glucosyltransferases were tested, GBP1 displayed the highest affinity for the glucan from the soluble-product, primer-independent glucosyltransferase.

The effects of the insulin-like growth factors and transforming growth factor beta on the Jun proto-oncogene family in MC3T3-E1 cells.

Previous studies have demonstrated that when cells of the mouse osteoblastic cell line MC3T3-E1 are exposed to IGF-I and IGF-II they exhibit rapid and transient induction of the transcript from the proto-oncogene c-fos [8]. To clarify the relationship between induction of cell proliferation and proto-oncogene expression in MC3T3-E1 cells, the acute affects of IGF-I and IGF-II, growth factors that stimulate cell proliferation, and of TGF-beta 1, which inhibits cell proliferation, northern analyses with cDNA-derived probes for the proto-oncogenes c-jun, jun-B, and jun-D were undertaken. Concurrent northern analyses with a probe for c-fos extended our previous results to include the effect of TGF-beta 1 on c-fos. IGF-I does not induce the c-jun, jun-B, or jun-D transcripts, the former and latter being produced at detectable levels constitutively. After 1 hour of exposure to IGF-II the c-jun transcript response ranges from onefold to 13-fold and the jun-D transcript response ranges around two-fold. After 1 hour of exposure to TGF-beta 1, the jun-B transcript response ranges from eightfold to 24-fold, the c-fos transcript response ranges between sixfold and sevenfold. The differences observed in the magnitude and kinetics of the induction provoked by these growth factors is consistent with the presence of a regulatory circuit acting through the Jun family members which may act to stimulate transcription differentially when bound to DNA either as homodimers or, with Fos family proteins, as heterodimers.

Sequence comparison and predicted structure for the four exon-encoded regions of human insulin-like growth factor binding protein 4.

The IGFBPs bind to and modulate the function of the IGFs in various ways. Human IGFBP-4 inhibits IGF mediated cell proliferation. The IGFBP exon-encoded regions were aligned and secondary structure predictions for hIGFBP-4 were developed yielding predicted 3D co-ordinates for each such region of hIGFBP-4. The exon 1 encoded region is the most conserved among the IGFBPs. That of hIGFBP-4 is predicted as an array of beta-strands that include the glycine and cysteine rich IGFBP consensus pattern and that terminate with a helix. The exon 2 encoded region is the most variable among the IGFBPs. That of hIGFBP-4 is predicted as mostly an amphipathic helix. The remaining regions are also conserved among the IGFBPs. Those of hIGFBP-4 are also predicted to contain helices. The predicted structure of hIGFBP-4 comprises amino terminal beta-strands with four helices in the carboxy terminal two thirds of the molecule.

Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding.

The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.