Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.04) nM, which is comparable to the dissociation constant of insulin from its receptor in hepatocytes. We used this assay to examine possible residual insulin-like activity of the four major fragments formed upon insulin degradation by insulin protease. Fragments A1-13B1-9, A1-14B1-9,and A14-21B14-30 showed no measurable activity. Although preparations of fragment A14-21B10-30 displayed dose-dependent agonist activity with an EC50 of 380 (+/-40) nM, we conclude that this was due to an insulin-like impurity since the chemically synthesized fragment showed no such activity. In summary, this bioassay demonstrates the action of insulin on glycogen formation in hepatocytes and provides a rapid and sensitive measurement of insulin-like activity which could facilitate screening studies.

Development of an isotope dilution assay for precise determination of insulin, C-peptide, and proinsulin levels in non-diabetic and type II diabetic individuals with comparison to immunoassay.

We describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (+/- 2) to 92 (+/- 8), 335 (+/- 11) to 821 (+/- 24), and 6 (+/- 1) to 37 (+/- 3) pM, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination.

A stable isotope dilution assay for the in vivo determination of insulin levels in humans by mass spectrometry.

Insulin levels in humans were measured by a new assay, the isotope dilution assay (IDA), based on stable isotope dilution mass spectrometry. A known amount of a deuterated analog of insulin was used as an internal standard and added to the serum samples before sample processing. After isolation by immunoaffinity chromatography and solid phase extraction, followed by a purification step on reversed-phase microbore high-performance liquid chromatography (HPLC), the insulin-containing fraction was analyzed by mass spectrometry. The relative intensity of the signals due to insulin and its deuterated analog in the mass spectrum was used to determine the concentration of insulin in the sample. Using serum samples of 0.5-2.0 ml, we were able to measure insulin levels in the range of 3-1700 pmol/l in several clinical samples from type II diabetic patients. The basal level of endogenous insulin was also determined in two normal subjects and found to be approximately 20 pmol/l. Insulin secretion was followed after the ingestion of 75 g glucose in one healthy volunteer. Finally, the determination of the insulin level of one hemolyzed post-mortem blood sample, for which immunoassays gave inconsistent results, was performed to help forensic investigations. Our results showed a good correlation with standard immunoassay data, except in six samples where much lower values were obtained by our stable isotope dilution assay, suggesting an overestimation of insulin levels by immunoassay in some cases. As it is not subject to immunological interferences by insulin-related compounds, this new assay has a major clinical advantage in that it avoids confusions related to hyperinsulinemia.

Analysis of the mechanism of assembly of cleaved barnase from two peptide fragments and its relevance to the folding pathway of uncleaved barnase.

A peptide corresponding to residues 1-22 of barnase that contains its major alpha-helix (residues 6-18) binds rapidly to the complementary peptide (residues 23-110) to form a catalytically active complex with near-native structure. Peptide 1-22 is approximately 3% helical in aqueous solution at 25 degrees C. A set of mutations in the helical regions of (1-22) cause the helix to be destabilized. We have investigated the mechanism of assembly of the peptides by analyzing the kinetics and equilibria of association of those mutants of (1-22) with native (23-110). The association reaction follows second-order kinetics. Virtually all the change in stability of the complex on mutation is reflected in changes in the association rate constant with the dissociation rate constant being very little affected. Both Brønsted and theta-value analyses show that the helix is essentially fully formed in the transition state for the association at all the positions probed (residues 13-18). Peptide (23-110) contains all the residues necessary for catalysis. The complexes between all mutants peptides (1-22) with (23-110) are all only 10% active, however. The noncovalent complex is destabilized less by mutations in the helix than is the intact protein. Double-mutant cycle and other analyses show, however, that the intrahelical interactions are as strong in the noncovalent complex as in the intact protein and so the interactions between the helix and the rest of the protein must be weakened on cleavage of the 22-23 bond. This could well lead to effects on catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies on peptides corresponding to mutants of the major alpha-helix of barnase.

The structures of a family of peptides that contain variants of the major alpha-helix of barnase (residues 6-18) have been analyzed in solution by circular dichroism (CD) and 1H NMR at various concentrations of the helix-inducing cosolvent trifluoroethanol (TFE). The very low equilibrium constant (approximately 10(-2) for the formation of helix in water KH2O was estimated from titration of the helical ellipticity signal at 222 nm with [TFE] using the equation, KTFE = KH2O exp((m/RT) [TFE]/[H2O]), where KTFE is the equilibrium constant for the formation of helix in TFE/H2O and m is characteristic for each peptide but appears to be proportional to the lengths of related helices. NMR studies show that the peptide is mainly random coil in water, but that the helix is induced cooperatively by TFE and extends from residues 6 to 18 for wild-type peptide in 35% TFE. The mutant peptide Tyr-17-->Ala, however, has a helical region extending only for residues 9-15. Truncation of the helix upon mutation is also detected in the TFE titration procedure, which finds correspondingly lowered m-values upon mutation. This is is also supported by measurements of the pH dependence of KH2O, which is caused by the ionization of the C-cap residue, His-18, whose pKa is raised by the interaction of the protonated form with the helix dipole. Whereas there is an apparent charge/dipole interaction energy of 1.1 kcal mol-1 in the wild-type peptide, similar to that measured in the native protein, this drops dramatically upon mutations that disrupt the C-terminus of the helix. Mutation of Tyr-17-->Ala lowers KH2O only slightly, as do the other helix-destabilizing mutations. The combined results show that the helix-weakening effects of mutations act here primarily by shortening the length of the helix, with smaller effects on the equilibrium constants between helix and coil (KH2O).(ABSTRACT TRUNCATED AT 250 WORDS)

Folding of barnase in parts.

Stretches of residual structure in the unfolded states of proteins could possibly constitute crucial regions that initiate protein folding. We are searching for such regions in barnase by dividing it into fragments. By this means, we can search for regions that just form within local sequences. We are also employing methods that can detect low levels of residual structure. In this study, we examine the fragment 1-22 and a large fragment (23-110) that contains all of the catalytic residues. Fragment 1-22 contains the first alpha-helix, and fragment 23-110 contains the second alpha-helix and beta-sheet structure-forming residues of native barnase. These fragments bind together rapidly and tightly upon association to form a fully native-like complex. Studies by circular dichroism and fluorescence spectroscopy indicate that each fragment is mainly disordered. However, we find by a procedure of titration with trifluoroethanol that about 3% of fragment 1-22 is helical in water at 25 degrees C. Importantly, we have detected residual catalytic activity in fragment 23-110 toward GpUp and RNA and the ability to bind the polypeptide inhibitor of barnase, barstar, suggesting that this fragment can form a native-like conformation in water. The catalytic activity does not result from a small amount of contaminating impurity of parent enzyme or other ribonuclease, since the activity requires a 1:1 mole ratio of fragment to barstar for complete inhibition, and the activity is lost in much lower concentrations of urea than are required to denature the parent enzyme. There is a very weak signal in the near-UV CD spectrum of the large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)