We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.

Native somatomedin (SM) has an apparent mol wt of at least 60,000 daltons, while all the purified SM peptides have mol wt 5-8,000 daltsons. We have studied the behavior of somatomedin in plasma during gel filtration at pH 8.1 and pH 2.8, and by the recombination of fractions. Using porcine bioassay for SM, displacement of 125I-insulin by SM, and binding of 125I-SM to plasma proteins, we can demonstrate that the disparity in apparent mol wt of SM is due to reversible binding to plasma proteins which are themselves biologically inactive. This binding site is specific for the SM peptides, saturable, and has high affinity and relatively low capacity.

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The somatomedin (SM) peptides are carried in plasma complexed to specific SM-binding proteins (SMBPs). In addition to the SMBP in the complex, there is an unsaturated SMBP in plasma in which the SMBP is not occupied by SMs. We have compared levels of unsaturated SMBP in 7 normal adults to those in 21 children with GH deficiency before and during treatment with hGH (0.1 U/kg.day) for 4 days. In addition, the SM-C/insulin-like growth factor I (IGF-I) content of each plasma was measured by RIA. There was a significant (P less than 0.01) difference in unsaturated SMBP levels between normal controls (17.8 +/- 0.8% bound/20 microliters) and untreated hypopituitary patients (27.8 +/- 2.2% bound/20 microliters). Thus, a lower SM-C/IGF-I content was associated with a higher unsaturated SMBP level. Furthermore, there was a significant negative correlation between SM-C/IGF-I content and unsaturated SMBP in untreated hypopituitary patients (r = 0.73; P less than 0.0001). Treatment with hGH normalized the mean unsaturated SMBP level in hypopituitary patients within 2 days. Full displacement curves and Scatchard analysis showed that the increased unsaturated SMBP level in hypopituitary plasma was entirely due to an increased affinity (8.4 X 0.9 X 10(-10) M) compared to normal (2.3 X 0.2 X 10(-9) M). A higher affinity form of unsaturated SMBP is uniquely present in hypopituitarism and disappears with hGH treatment. The measurement of this unsaturated SMBP mirrors the SM peptide content.